Whole-cell organelle segmentation in volume electron microscopy.

Cells contain hundreds of organelles and macromolecular assemblies. Obtaining a complete understanding of their intricate organization requires the nanometre-level, three-dimensional reconstruction of whole cells, which is only feasible with robust and scalable automatic methods. Here, to support the development of such methods, we annotated up to 35 different cellular organelle classes-ranging from endoplasmic reticulum to microtubules to ribosomes-in diverse sample volumes from multiple cell types imaged at a near-isotropic resolution of 4 nm per voxel with focused ion beam scanning electron microscopy (FIB-SEM)1. We trained deep learning architectures to segment these structures in 4 nm and 8 nm per voxel FIB-SEM volumes, validated their performance and showed that automatic reconstructions can be used to directly quantify previously inaccessible metrics including spatial interactions between cellular components. We also show that such reconstructions can be used to automatically register light and electron microscopy images for correlative studies. We have created an open data and open-source web repository, 'OpenOrganelle', to share the data, computer code and trained models, which will enable scientists everywhere to query and further improve automatic reconstruction of these datasets.

The impact of smoking cessation attempts on stress levels.

BACKGROUND: Cigarette smoking is a major health risk, particularly in male South Koreans. Smoking cessation can benefit health; however, the process of quitting smoking is difficult to some smokers and shows its relationship to their stress level. The hypothesis of this study is that who has failed attempts to stop smoking induce more stress than habitual smoking. METHODS: To test this, the analysis on the association between smoking cessation attempts and stress levels in smokers was performed. The Korean Community Health Survey (2011-2016) data with the total of 488,417 participants' data were used for this study. Survey data were analyzed using the chi-square test and logistic regression. As the dependent variable, self-reported level of stress was selected. RESULTS: Of the subject population, 78.3% (63.3% males, 81.4% females) felt stressed. Among participants who successfully stopped smoking, 73.0% (72.6% males, 78.1% females) reported feeling stressed. In contrast, of those who failed to stop smoking, 83.3% (83.6% males, 86.3% females) reported high stress levels. Among those who did not attempt smoking cessation, 81.1% (81.2% males, 80.3% females) responded that they experienced stress. Those who failed to stop smoking had higher odds of stress than those who did not attempt smoking cessation [odds ratio (OR) 1.11, 95% confidence interval (CI) 1.09-1.14, p < 0.001]. Those who successfully stopped smoking had lower odds of stress than those who did not attempt smoking cessation (OR 0.87, 95% CI 0.86-0.89, p < 0.001). CONCLUSION: The study found an association between unsuccessful smoking cessation and stress level. As the result, people who failed smoking cessation showed higher stress. These data should be considered in health policy recommendations for smokers.

Capillarity Guided Patterning of Microliquids.

Soft lithography and other techniques have been developed to investigate biological and chemical phenomena as an alternative to photolithography-based patterning methods that have compatibility problems. Here, a simple approach for nonlithographic patterning of liquids and gels inside microchannels is described. Using a design that incorporates strategically placed microstructures inside the channel, microliquids or gels can be spontaneously trapped and patterned when the channel is drained. The ability to form microscale patterns inside microfluidic channels using simple fluid drain motion offers many advantages. This method is geometrically analyzed based on hydrodynamics and verified with simulation and experiments. Various materials (i.e., water, hydrogels, and other liquids) are successfully patterned with complex shapes that are isolated from each other. Multiple cell types are patterned within the gels. Capillarity guided patterning (CGP) is fast, simple, and robust. It is not limited by pattern shape, size, cell type, and material. In a simple three-step process, a 3D cancer model that mimics cell-cell and cell-extracellular matrix interactions is engineered. The simplicity and robustness of the CGP will be attractive for developing novel in vitro models of organ-on-a-chip and other biological experimental platforms amenable to long-term observation of dynamic events using advanced imaging and analytical techniques.

Free-standing gold-nanoparticle monolayer film fabricated by protein self-assembly of α-synuclein.

Free-standing nanoparticle films are of great importance for developing future nano-electronic devices. We introduce a protein-based fabrication strategy of free-standing nanoparticle monolayer films. α-Synuclein, an amyloidogenic protein, was utilized to yield a tightly packed gold-nanoparticle monolayer film interconnected by protein ß-sheet interactions. Owing to the stable protein-protein interaction, the film was successfully expanded to a 4-inch diameter sheet, which has not been achieved with any other free-standing nanoparticle monolayers. The film was flexible in solution, so it formed a conformal contact, surrounding even microspheres. Additionally, the monolayer film was readily patterned at micrometer-scale and thus unprecedented double-component nanoparticle films were fabricated. Therefore, the free-floating gold-nanoparticle monolayer sheets with these properties could make the film useful for the development of bio-integrated nano-devices and high-performance sensors.

Microscale Flow Control and Droplet Generation Using Arduino-Based Pneumatically-Controlled Microfluidic Device.

Microfluidics are crucial for managing small-volume analytical solutions for various applications, such as disease diagnostics, drug efficacy testing, chemical analysis, and water quality monitoring. The precise control of flow control devices can generate diverse flow patterns using pneumatic control with solenoid valves and a microcontroller. This system enables the active modulation of the pneumatic pressure through Arduino programming of the solenoid valves connected to the pressure source. Additionally, the incorporation of solenoid valve sets allows for multichannel control, enabling simultaneous creation and manipulation of various microflows at a low cost. The proposed microfluidic flow controller facilitates accurate flow regulation, especially through periodic flow modulation beneficial for droplet generation and continuous production of microdroplets of different sizes. Overall, we expect the proposed microfluidic flow controller to drive innovative advancements in technology and medicine owing to its engineering precision and versatility.

Transparent Intracellular Sensing Platform with Si Needles for Simultaneous Live Imaging.

Vertically ordered Si needles are of particular interest for long-term intracellular recording owing to their capacity to infiltrate living cells with negligible damage and minimal toxicity. Such intracellular recordings could greatly benefit from simultaneous live cell imaging without disrupting their culture, contributing to an in-depth understanding of cellular function and activity. However, the use of standard live imaging techniques, such as inverted and confocal microscopy, is currently impeded by the opacity of Si wafers, typically employed for fabricating vertical Si needles. Here, we introduce a transparent intracellular sensing platform that combines vertical Si needles with a percolated network of Au-Ag nanowires on a transparent elastomeric substrate. This sensing platform meets all prerequisites for simultaneous intracellular recording and imaging, including electrochemical impedance, optical transparency, mechanical compliance, and cell viability. Proof-of-concept demonstrations of this sensing platform include monitoring electrical potentials in cardiomyocyte cells and in three-dimensionally engineered cardiovascular tissue, all while conducting live imaging with inverted and confocal microscopes. This sensing platform holds wide-ranging potential applications for intracellular research across various disciplines such as neuroscience, cardiology, muscle physiology, and drug screening.

Architecture and dynamics of a desmosome-endoplasmic reticulum complex.

The endoplasmic reticulum (ER) forms a dynamic network that contacts other cellular membranes to regulate stress responses, calcium signalling and lipid transfer. Here, using high-resolution volume electron microscopy, we find that the ER forms a previously unknown association with keratin intermediate filaments and desmosomal cell-cell junctions. Peripheral ER assembles into mirror image-like arrangements at desmosomes and exhibits nanometre proximity to keratin filaments and the desmosome cytoplasmic plaque. ER tubules exhibit stable associations with desmosomes, and perturbation of desmosomes or keratin filaments alters ER organization, mobility and expression of ER stress transcripts. These findings indicate that desmosomes and the keratin cytoskeleton regulate the distribution, function and dynamics of the ER network. Overall, this study reveals a previously unknown subcellular architecture defined by the structural integration of ER tubules with an epithelial intercellular junction.

Biodegradable silicon nanoneedles for ocular drug delivery.

Ocular drug delivery remains a grand challenge due to the complex structure of the eye. Here, we introduce a unique platform of ocular drug delivery through the integration of silicon nanoneedles with a tear-soluble contact lens. The silicon nanoneedles can penetrate into the cornea in a minimally invasive manner and then undergo gradual degradation over the course of months, enabling painless and long-term sustained delivery of ocular drugs. The tear-soluble contact lens can fit a variety of corneal sizes and then quickly dissolve in tear fluid within a minute, enabling an initial burst release of anti-inflammatory drugs. We demonstrated the utility of this platform in effectively treating a chronic ocular disease, such as corneal neovascularization, in a rabbit model without showing a notable side effect over current standard therapies. This platform could also be useful in treating other chronic ocular diseases.

Smart soft contact lenses for continuous 24-hour monitoring of intraocular pressure in glaucoma care.

Continuous monitoring of intraocular pressure, particularly during sleep, remains a grand challenge in glaucoma care. Here we introduce a class of smart soft contact lenses, enabling the continuous 24-hour monitoring of intraocular pressure, even during sleep. Uniquely, the smart soft contact lenses are built upon various commercial brands of soft contact lenses without altering their intrinsic properties such as lens power, biocompatibility, softness, transparency, wettability, oxygen transmissibility, and overnight wearability. We show that the smart soft contact lenses can seamlessly fit across different corneal curvatures and thicknesses in human eyes and therefore accurately measure absolute intraocular pressure under ambulatory conditions. We perform a comprehensive set of in vivo evaluations in rabbit, dog, and human eyes from normal to hypertension to confirm the superior measurement accuracy, within-subject repeatability, and user comfort of the smart soft contact lenses beyond current wearable ocular tonometers. We envision that the smart soft contact lenses will be effective in glaucoma care.

All-printed stretchable corneal sensor on soft contact lenses for noninvasive and painless ocular electrodiagnosis.

Electroretinogram examinations serve as routine clinical procedures in ophthalmology for the diagnosis and management of many ocular diseases. However, the rigid form factor of current corneal sensors produces a mismatch with the soft, curvilinear, and exceptionally sensitive human cornea, which typically requires the use of topical anesthesia and a speculum for pain management and safety. Here we report a design of an all-printed stretchable corneal sensor built on commercially-available disposable soft contact lenses that can intimately and non-invasively interface with the corneal surface of human eyes. The corneal sensor is integrated with soft contact lenses via an electrochemical anchoring mechanism in a seamless manner that ensures its mechanical and chemical reliability. Thus, the resulting device enables the high-fidelity recording of full-field electroretinogram signals in human eyes without the need of topical anesthesia or a speculum. The device, superior to clinical standards in terms of signal quality and comfortability, is expected to address unmet clinical needs in the field of ocular electrodiagnosis.

Rapid custom prototyping of soft poroelastic biosensor for simultaneous epicardial recording and imaging.

The growing need for the implementation of stretchable biosensors in the body has driven rapid prototyping schemes through the direct ink writing of multidimensional functional architectures. Recent approaches employ biocompatible inks that are dispensable through an automated nozzle injection system. However, their application in medical practices remains challenged in reliable recording due to their viscoelastic nature that yields mechanical and electrical hysteresis under periodic large strains. Herein, we report sponge-like poroelastic silicone composites adaptable for high-precision direct writing of custom-designed stretchable biosensors, which are soft and insensitive to strains. Their unique structural properties yield a robust coupling to living tissues, enabling high-fidelity recording of spatiotemporal electrophysiological activity and real-time ultrasound imaging for visual feedback. In vivo evaluations of custom-fit biosensors in a murine acute myocardial infarction model demonstrate a potential clinical utility in the simultaneous intraoperative recording and imaging on the epicardium, which may guide definitive surgical treatments.

Fractal Web Design of a Hemispherical Photodetector Array with Organic-Dye-Sensitized Graphene Hybrid Composites.

The vision system of arthropods consists of a dense array of individual photodetecting elements across a curvilinear surface. This compound-eye architecture could be a useful model for optoelectronic sensing devices that require a large field of view and high sensitivity to motion. Strategies that aim to mimic the compound-eye architecture involve integrating photodetector pixels with a curved microlens, but their fabrication on a curvilinear surface is challenged by the use of standard microfabrication processes that are traditionally designed for planar, rigid substrates (e.g., Si wafers). Here, a fractal web design of a hemispherical photodetector array that contains an organic-dye-sensitized graphene hybrid composite is reported to serve as an effective photoactive component with enhanced light-absorbing capabilities. The device is first fabricated on a planar Si wafer at the microscale and then transferred to transparent hemispherical domes with different curvatures in a deterministic manner. The unique structural property of the fractal web design provides protection of the device from damage by effectively tolerating various external loads. Comprehensive experimental and computational studies reveal the essential design features and optoelectronic properties of the device, followed by the evaluation of its utility in the measurement of both the direction and intensity of incident light.

Bioresorbable, Miniaturized Porous Silicon Needles on a Flexible Water-Soluble Backing for Unobtrusive, Sustained Delivery of Chemotherapy.

Conventional melanoma therapies suffer from the toxicity and side effects of repeated treatments due to the aggressive and recurrent nature of melanoma cells. Less-invasive topical chemotherapies by utilizing polymeric microneedles have emerged as an alternative, but the sustained, long-lasting release of drug cargos remains challenging. In addition, the size of the microneedles is relatively bulky for the small, curvilinear, and exceptionally sensitive cornea for the treatment of ocular melanoma. Here, we report a design of bioresorbable, miniaturized porous-silicon (p-Si) needles with covalently linked drug cargos at doses comparable to those of conventional polymeric microneedles. The p-Si needles are built on a water-soluble film as a temporary flexible holder that can be intimately interfaced with the irregular surface of living tissues, followed by complete dissolution with saline solution within 1 min. Consequently, the p-Si needles remain embedded inside tissues and then undergo gradual degradation, allowing for sustained release of the drug cargos. Its utility in unobtrusive topical delivery of chemotherapy with minimal side effects is demonstrated in a murine melanoma model.

Microstructure guided multi-scale liquid patterning on an open surface.

Liquid patterning is a quintessential aspect in cell-based screening. While there are a variety of methods to handle microliquids utilizing surface treatments, complex microfluidic systems, and automated dispensing, most of the stated methods are both expensive and difficult to implement. Here, we present a fast multi-scale microliquid-patterning method on an open surface using embossed microstructures without surface modification. Arrays of micropillars can trap microliquids when a bulk drop is swept by an elastic sweeper on polystyrene (PS) substrates. The patterning mechanism on a basic form of a 2 × 2 rectangular array of circular pillars is analyzed theoretically and verified with experiments. Nanoliter-to-microliter volumes of liquids are patterned into various shapes by arranging the pillars based on the analysis. Furthermore, an array of geometrically modified pillars can capture approximately 8000 droplets on a large substrate (55 mm × 55 mm) in one step. Given the simplistic method of wipe patterning, the proposed platform can be utilized in both manual benchtop and automated settings. We will provide proof of concept experiments of single colony isolation using nanoliter-scale liquid patterning and of human angiogenic vessel formation using sequential patterning of microliter-scale liquids.

Antimycin A induces apoptosis in As4.1 juxtaglomerular cells.

Antimycin A, an inhibitor of electron transport in mitochondria, has been used as reactive oxygen species (ROS) generator in the biological system. Here, we investigated the in vitro effect of antimycin A on apoptosis in As4.1 juxtaglomerular cells. Antimycin A efficiently induced apoptosis in As4.1 cells as evidenced by flow cytometric detection of sub-G(1) DNA content, annexin V binding assay and DAPI staining. This apoptotic process was accompanied by loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bcl-2 decrease, caspase-3 activation and PARP cleavage. All of caspase inhibitors tested in this experiment failed to rescue As4.1 cells from antimycin A-induced cell death at the time of 48 h in view of sub-G(1) cells and annexin V positive staining cells. However, with regard to the mitochondrial membrane potential (DeltaPsi(m)), pan caspase inhibitor (Z-VAD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK) at the concentration of 25 microM noticeably decreased the loss of mitochondrial membrane potential (DeltaPsi(m)) in antimycin A-treated cells. Taken together, we have demonstrated that antimycin A as an inhibitor of electron transport in mitochondria potently induces apoptosis in As4.1 juxtaglomerular cells.

A microfluidic platform for quantitative analysis of cancer angiogenesis and intravasation.

Understanding the mechanism behind cancer metastasis is a major challenge in cancer biology. Several in vitro models have been developed to mimic a cancer microenvironment by engineering cancer-endothelial cell (EC) and cancer-stromal cell interactions. It has been challenging to realistically mimic angiogenesis, intravasation, and extravasation using macro-scale approaches but recent progress in microfluidics technology has begun to yield promising results. We present a metastasis chip that produce microvessels, where EC and stromal cells can be patterned in close proximity to tumor cells. The vessels are formed following a natural morphogenic process and have smooth boundaries with proper cell-cell junctions. The engineered microvessels are perfusable and have well-defined openings toward inlet and outlet channels. The ability to introduce cancer cells into different locations bordering to the microvessel wall allowed generation and maintenance of appropriate spatial gradients of growth factors and attractants. Cancer angiogenesis and its inhibition by anti-vascular endothelial growth factor (bevacizumab) treatment were successfully reproduced in the metastasis chip. Cancer intravasation and its modulation by treatment of tumor necrosis factor-α were also modeled. Compared to other models, the unique design of the metastasis chip that engineers a clear EC-cancer interface allows precise imaging and quantification of angiogenic response as well as tumor cell trans-endothelial migration. The metastasis chip presented here has potential applications in the investigation of fundamental cancer biology as well as in drug screening.

Improved severe hepatopulmonary syndrome after liver transplantation in an adolescent with end-stage liver disease secondary to biliary atresia.

Hepatopulmonary syndrome (HPS) is a serious complication of end-stage liver disease, which is characterized by hypoxia, intrapulmonary vascular dilatation, and liver cirrhosis. Liver transplantation (LT) is the only curative treatment modality for patients with HPS. However, morbidity and mortality after LT, especially in cases of severe HPS, remain high. This case report describes a patient with typical findings of an extracardiac pulmonary arteriovenous shunt on contrast-enhanced transesophageal echocardiography (TEE), and clubbing fingers, who had complete correction of HPS by deceased donor LT. The patient was a 16-year-old female who was born with biliary atresia and underwent porto-enterostomy on the 55th day after birth. She had been suffered from progressive liver failure with dyspnea, clubbing fingers, and cyanosis. Preoperative arterial blood gas analysis revealed severe hypoxia (arterial O2 tension of 54.5 mmHg and O2 saturation of 84.2%). Contrast-enhanced TEE revealed an extracardiac right-to-left shunt, which suggested an intrapulmonary arteriovenous shunt. The patient recovered successfully after LT, not only with respect to physical parameters but also for pychosocial activity, including school performance, during the 30-month follow-up period.