Seq-Scope Protocol: Repurposing Illumina Sequencing Flow Cells for High-Resolution Spatial Transcriptomics.

Spatial transcriptomics (ST) technologies represent a significant advance in gene expression studies, aiming to profile the entire transcriptome from a single histological slide. These techniques are designed to overcome the constraints faced by traditional methods such as immunostaining and RNA in situ hybridization, which are capable of analyzing only a few target genes simultaneously. However, the application of ST in histopathological analysis is also limited by several factors, including low resolution, a limited range of genes, scalability issues, high cost, and the need for sophisticated equipment and complex methodologies. Seq-Scope-a recently developed novel technology-repurposes the Illumina sequencing platform for high-resolution, high-content spatial transcriptome analysis, thereby overcoming these limitations. Here we provide a detailed step-by-step protocol to implement Seq-Scope with an Illumina NovaSeq 6000 sequencing flow cell that allows for the profiling of multiple tissue sections in an area of 7 mm × 7 mm or larger. In addition to detailing how to prepare a frozen tissue section for both histological imaging and sequencing library preparation, we provide comprehensive instructions and a streamlined computational pipeline to integrate histological and transcriptomic data for high-resolution spatial analysis. This includes the use of conventional software tools for single cell and spatial analysis, as well as our recently developed segmentation-free method for analyzing spatial data at submicrometer resolution. Given its adaptability across various biological tissues, Seq-Scope establishes itself as an invaluable tool for researchers in molecular biology and histology.

High-Resolution Spatial Transcriptomic Atlas of Mouse Soleus Muscle: Unveiling Single Cell and Subcellular Heterogeneity in Health and Denervation.

Skeletal muscle is essential for both movement and metabolic processes, characterized by a complex and ordered structure. Despite its importance, a detailed spatial map of gene expression within muscle tissue has been challenging to achieve due to the limitations of existing technologies, which struggle to provide high-resolution views. In this study, we leverage the Seq-Scope technique, an innovative method that allows for the observation of the entire transcriptome at an unprecedented submicron spatial resolution. By applying this technique to the mouse soleus muscle, we analyze and compare the gene expression profiles in both healthy conditions and following denervation, a process that mimics aspects of muscle aging. Our approach reveals detailed characteristics of muscle fibers, other cell types present within the muscle, and specific subcellular structures such as the postsynaptic nuclei at neuromuscular junctions, hybrid muscle fibers, and areas of localized expression of genes responsive to muscle injury, along with their histological context. The findings of this research significantly enhance our understanding of the diversity within the muscle cell transcriptome and its variation in response to denervation, a key factor in the decline of muscle function with age. This breakthrough in spatial transcriptomics not only deepens our knowledge of muscle biology but also sets the stage for the development of new therapeutic strategies aimed at mitigating the effects of aging on muscle health, thereby offering a more comprehensive insight into the mechanisms of muscle maintenance and degeneration in the context of aging and disease.

Tailoring MXene Thickness and Functionalization for Enhanced Room-Temperature Trace NO2 Sensing.

In this study, precise control over the thickness and termination of Ti3C2TX MXene flakes is achieved to enhance their electrical properties, environmental stability, and gas-sensing performance. Utilizing a hybrid method involving high-pressure processing, stirring, and immiscible solutions, sub-100 nm MXene flake thickness is achieved within the MXene film on the Si-wafer. Functionalization control is achieved by defunctionalizing MXene at 650 °C under vacuum and H2 gas in a CVD furnace, followed by refunctionalization with iodine and bromine vaporization from a bubbler attached to the CVD. Notably, the introduction of iodine, which has a larger atomic size, lower electronegativity, reduce shielding effect, and lower hydrophilicity (contact angle: 99°), profoundly affecting MXene. It improves the surface area (36.2 cm2 g-1), oxidation stability in aqueous/ambient environments (21 days/80 days), and film conductivity (749 S m-1). Additionally, it significantly enhances the gas-sensing performance, including the sensitivity (0.1119 Ω ppm-1), response (0.2% and 23% to 50 ppb and 200 ppm NO2), and response/recovery times (90/100 s). The reduced shielding effect of the -I-terminals and the metallic characteristics of MXene enhance the selectivity of I-MXene toward NO2. This approach paves the way for the development of stable and high-performance gas-sensing two-dimensional materials with promising prospects for future studies.

FICTURE: Scalable segmentation-free analysis of submicron resolution spatial transcriptomics.

Spatial transcriptomics (ST) technologies have advanced to enable transcriptome-wide gene expression analysis at submicron resolution over large areas. Analysis of high-resolution ST data relies heavily on image-based cell segmentation or gridding, which often fails in complex tissues due to diversity and irregularity of cell size and shape. Existing segmentation-free analysis methods scale only to small regions and a small number of genes, limiting their utility in high-throughput studies. Here we present FICTURE, a segmentation-free spatial factorization method that can handle transcriptome-wide data labeled with billions of submicron resolution spatial coordinates. FICTURE is orders of magnitude more efficient than existing methods and it is compatible with both sequencing- and imaging-based ST data. FICTURE reveals the microscopic ST architecture for challenging tissues, such as vascular, fibrotic, muscular, and lipid-laden areas in real data where previous methods failed. FICTURE's cross-platform generality, scalability, and precision make it a powerful tool for exploring high-resolution ST.

Wireless Inchworm-like Compact Soft Robot by Induction Heating of Magnetic Composite.

Microrobots and nanorobots have been produced with various nature-inspired soft materials and operating mechanisms. However, freely operating a wirelessly miniaturized soft robot remains a challenge. In this study, a wireless crawling compact soft robot using induction heating was developed. The magnetic composite heater built into the robot was heated wirelessly via induction heating, causing a phase change in the working fluid surrounding the heater. The pressure generated from the evaporated fluid induces the bending of the robot, which is composed of elastomers. During one cycle of bending by heating and shrinking by cooling, the difference in the frictional force between the two legs of the robot causes it to move forward. This robot moved 7240 µm, representing 103% of its body length, over nine repetitions. Because the robot's surface is made of biocompatible materials, it offers new possibilities for a soft exploratory microrobot that can be used inside a living body or in a narrow pipe.

Wireless Micro Soft Actuator without Payloads Using 3D Helical Coils.

To receive a greater power and to demonstrate the soft bellows-shaped actuator's wireless actuation, micro inductors were built for wireless power transfer and realized in a three-dimensional helical structure, which have previously been built in two-dimensional spiral structures. Although the three-dimensional helical inductor has the advantage of acquiring more magnetic flux linkage than the two-dimensional spiral inductor, the existing microfabrication technique produces a device on a two-dimensional plane, as it has a limit to building a complete three-dimensional structure. In this study, by using a three-dimensional printed soluble mold technique, a three-dimensional heater with helical coils, which have a larger heating area than a two-dimensional heater, was fabricated with three-dimensional receiving inductors for enhanced wireless power transfer. The three-dimensional heater connected to the three-dimensional helical inductor increased the temperature of the liquid and gas inside the bellows-shaped actuator while reaching 176.1% higher temperature than the heater connected to the two-dimensional spiral inductor. Thereby it enables a stroke of the actuator up to 522% longer than when it is connected to the spiral inductor. Therefore, three-dimensional micro coils can offer a significant approach to the development of wireless micro soft robots without incurring heavy and bulky parts such as batteries.

Combination of PD98059 and TGF-ß1 Efficiently Differentiates Human Urine-Derived Stem Cells into Smooth Muscle Cells.

Pluripotent adult stem cells have potential applications in cell therapy and tissue engineering. Urine-derived stem cells (UDSCs) differentiate into various cell types. Here, we attempted to differentiate human UDSCs (hUDSCs) into smooth muscle cells (SMCs) using transforming growth factor-beta 1 (TGF-ß1) and/or PD98059, an extracellular signal-regulated kinase (ERK) inhibitor. Both quantitative polymerase chain reaction (qPCR) and Western blot analysis showed that the expression of messenger ribonucleic acid (mRNA) and proteins for alpha-smooth muscle actin (α-SMA), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC), which are specific markers for SMCs, increased on day 9 after differentiation and again on day 14. The differentiated cells from human UDSCs (hUDSCs) with a combination of TGF-ß1 and PD98059 showed the highest expression of SMC marker proteins. Immunocytochemical staining performed to assess the molecular expression revealed CNN and α-SMA colocalizing in the cytoplasm. The cells that differentiated from hUDSCs with a combination of TGF-ß1 and PD98059 showed the strongest expression for CNN1, α-SMA, and SM-MHC. Functional testing of the differentiated cells revealed a stronger contractile capacity for the cells differentiated with a combination of PD98059 and TGF-ß1 than those differentiated with a single factor. These results suggest the combination of PD98059 and TGF-ß1 to be a more effective differentiation method and that differentiated SMCs could be used for restoring the functions of the sphincter muscle or bladder.

Thermopneumatic Soft Micro Bellows Actuator for Standalone Operation.

Typical pneumatic soft micro actuators can be manufactured without using heavy driving components such as pumps and power supplies by adopting an independent battery-powered mechanism. In this study, a thermopneumatically operated soft micro bellows actuator was manufactured, and the standalone operation of the actuator was experimentally validated. Thermopneumatic actuation is based on heating a sealed cavity inside the elastomer of the actuator to raise the pressure, leading to deflection of the elastomer. The bellows actuator was fabricated by casting polydimethylsiloxane (PDMS) using the 3D-printed soluble mold technique to prevent leakage, which is inherent in conventional soft lithography due to the bonding of individual layers. The heater, manufactured separately using winding copper wire, was inserted into the cavity of the bellows actuator, which together formed the thermopneumatic actuator. The 3D coil heater and bellows allowed immediate heat transfer and free movement in the intended direction, which is unachievable for conventional microfabrication. The fabricated actuator produced a stroke of 2184 µm, equivalent to 62% of the body, and exerted a force of 90.2 mN at a voltage of 0.55 V. A system in which the thermopneumatic actuator was driven by alkaline batteries and a control circuit also demonstrated a repetitive standalone operation.

Diphlorethohydroxycarmalol Attenuates Palmitate-Induced Hepatic Lipogenesis and Inflammation.

Non-alcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease, encompassing a range of conditions caused by lipid deposition within liver cells, and is also associated with obesity and metabolic diseases. Here, we investigated the protective effects of diphlorethohydroxycarmalol (DPHC), which is a polyphenol isolated from an edible seaweed, Ishige okamurae, on palmitate-induced lipotoxicity in the liver. DPHC treatment repressed palmitate-induced cytotoxicity, triglyceride content, and lipid accumulation. DPHC prevented palmitate-induced mRNA and protein expression of SREBP (sterol regulatory element-binding protein) 1, C/EBP (CCAAT-enhancer-binding protein) α, ChREBP (carbohydrate-responsive element-binding protein), and FAS (fatty acid synthase). In addition, palmitate treatment reduced the expression levels of phosphorylated AMP-activated protein kinase (AMPK) and sirtuin (SIRT)1 proteins, and DPHC treatment rescued this reduction. Moreover, DPHC protected palmitate-induced liver toxicity and lipogenesis, as well as inflammation, and enhanced AMPK and SIRT1 signaling in zebrafish. These results suggest that DPHC possesses protective effects against palmitate-induced toxicity in the liver by preventing lipogenesis and inflammation. DPHC could be used as a potential therapeutic or preventive agent for fatty liver diseases.

Direct differentiation of insulin-producing cells from human urine-derived stem cells.

The loss of pancreatic ß-cells is a cause of diabetes. Therefore, replacement of pancreatic ß-cells is a logical strategy for the treatment of diabetes, and the generation of insulin-producing cells (IPCs) from stem cells has been widely investigated as an alternative source for pancreatic ß-cells. Here, we isolated stem cells from human urine and investigated their differentiation potential into IPCs. We checked the expression of surface stem cell markers and stem cell transcription factors, and found that the isolated human urine-derived stem cells (hUDSCs) expressed the stem cell markers CD44, CD90, CD105 and stage-specific embryonic antigen (SSEA)-4. In addition, these cells expressed octamer binding transcription factor (Oct)4 and vimentin. hUDSCs could differentiate into adipocytes and osteocytes, as evidenced by Oil-red O staining and Alizarin Red S-staining of differentiated cells, respectively. When we directly differentiated hUDSCs into IPCs, the differentiated cells expressed mRNA for pancreatic transcription factors such as neurogenin (Ngn)3 and pancreatic and duodenal homeobox (Pdx)1. Differentiated IPCs expressed insulin and glucagon mRNA and protein, and these IPCs also secreted insulin in response to glucose stimulation. In conclusion, we found that hUDSCs can be directly differentiated into IPCs, which secrete insulin in response to glucose.

Indole-4-carboxaldehyde Isolated from Seaweed, Sargassum thunbergii, Attenuates Methylglyoxal-Induced Hepatic Inflammation.

Glucose degradation is aberrantly increased in hyperglycemia, which causes various harmful effects on the liver. Glyoxalase-1 (Glo-1) is a ubiquitous cellular enzyme that participates in the detoxification of methylglyoxal (MGO), a cytotoxic byproduct of glycolysis that induces protein modification (advanced glycation end-products, AGEs) and inflammation. Here, we investigated the anti-inflammatory effect of indole-4-carboxaldehyde (ST-I4C), which was isolated from the edible seaweed Sargassum thunbergii, on MGO-induced inflammation in HepG2 cells, a human hepatocyte cell line. ST-I4C attenuated the MGO-induced expression of inflammatory-related genes, such as tumor necrosis factor (TNF)-α and IFN-γ by activating nuclear factor-kappa B (NF-κB) without toxicity in HepG2 cells. In addition, ST-I4C reduced the MGO-induced AGE formation and the expression of the receptor for AGE (RAGE). Interestingly, both the mRNA and protein expression levels of Glo-1 increased following ST-I4C treatment, and the decrease in Glo-1 mRNA expression caused by MGO exposure was rescued by ST-I4C pretreatment. These results suggest that ST-I4C shows anti-inflammatory activity against MGO-induced inflammation in human hepatocytes by preventing an increase in the pro-inflammatory gene expression and AGE formation. Therefore, it represents a potential therapeutic agent for the prevention of hepatic steatosis.

Effects of Individuality, Education, and Image on Visual Attention: Analyzing Eye-tracking Data using Machine Learning.

Machine learning, particularly classification algorithms, constructs mathematical models from labeled data that can predict labels for new data. Using its capability to identify distinguishing patterns among multi-dimensional data, we investigated the impact of three factors on the observation of architectural scenes: individuality, education, and image stimuli. An analysis of the eye-tracking data revealed that (1) a velocity histogram was unique to individuals, (2) students of architecture and other disciplines could be distinguished via endogenous parameters, but (3) they were more distinct in terms of seeking structural versus symbolic elements. Because of the reverse nature of the classification algorithms that automatically learn from data, we could identify relevant parameters and distinguishing eye-tracking patterns that have not been reported in previous studies.

Polysiphonia japonica Extract Attenuates Palmitate-Induced Toxicity and Enhances Insulin Secretion in Pancreatic Beta-Cells.

Beta-cell loss is a major cause of the pathogenesis of diabetes. Elevated levels of free fatty acids may contribute to the loss of ß-cells. Using a transgenic zebrafish, we screened ~50 seaweed crude extracts to identify materials that protect ß-cells from free fatty acid damage. We found that an extract of the red seaweed Polysiphonia japonica (PJE) had a ß-cell protective effect. We examined the protective effect of PJE on palmitate-induced damage in ß-cells. PJE was found to preserve cell viability and glucose-induced insulin secretion in a pancreatic ß-cell line, Ins-1, treated with palmitate. Additionally, PJE prevented palmitate-induced insulin secretion dysfunction in zebrafish embryos and mouse primary islets and improved insulin secretion in ß-cells against palmitate treatment. These findings suggest that PJE protects pancreatic ß-cells from palmitate-induced damage. PJE may be a potential therapeutic functional food for diabetes.

Diphlorethohydroxycarmalol Attenuates Methylglyoxal-Induced Oxidative Stress and Advanced Glycation End Product Formation in Human Kidney Cells.

Diabetic nephropathy is the leading cause of end-stage renal disease in patients with diabetes mellitus. Oxidative stress has been shown to play an important role in pathogeneses of renal damage in diabetic patients. Here, we investigated the protective effect of diphlorethohydroxycarmalol (DPHC), which is a polyphenol isolated from an edible seaweed, Ishige okamurae, on methylglyoxal-induced oxidative stress in HEK cells, a human embryonic kidney cell line. DPHC treatment inhibited methylglyoxal- (MGO-) induced cytotoxicity and ROS production. DPHC activated the Nrf2 transcription factor and increased the mRNA expression of antioxidant and detoxification enzymes, consequently reducing MGO-induced advanced glycation end product formation. In addition, DPHC increased glyoxalase-1 mRNA expression and attenuated MGO-induced advanced glycation end product formation in HEK cells. These results suggest that DPHC possesses a protective activity against MGO-induced cytotoxicity in human kidney cells by preventing oxidative stress and advanced glycation end product formation. Therefore, it could be used as a potential therapeutic agent for the prevention of diabetic nephropathy.

Palmitate induces nitric oxide production and inflammatory cytokine expression in zebrafish.

Inflammation markers in zebrafish embryos reflect a toxic response that is common to other animal models and humans. Free fatty acids (FFAs) are known to cause damage in various tissues by inducing inflammation. In this study, we investigated whether a FFA (palmitate) induces inflammation in zebrafish embryos. Nitrous oxide (NO) production and cyclooxygenase-2 (COX-2) mRNA expression were increased in palmitate-treated zebrafish embryos in a dose-dependent manner. mRNA expression of pro-inflammatory cytokines, interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF- α), were also increased. Additionally, the mRNA expression of p65 nuclear factor-kB and I-kB-α were significantly increased after palmitate-treatment. Increased reactive oxygen species (ROS) expression was observed in palmitate-treated zebrafish embryos as well as pericardial edema. Additionally, mRNA expression of pro-inflammatory cytokines were increased in zebrafish liver and pancreas fed with palmitate-contained diet. Taken together, these results indicated that palmitate increases pro-inflammatory mediators in zebrafish embryos, suggesting that zebrafish could be an alternative animal model for inflammatory disease including diabetes.

Fabrication of truly 3D microfluidic channel using 3D-printed soluble mold.

The field of complex microfluidic channels is rapidly expanding toward channels with variable cross-sections (i.e., beyond simple rounded channels with a constant diameter), as well as channels whose trajectory can be outside of a single plane. This paper introduces the use of three-dimensional (3D) printed soluble wax as cast molds for rapid fabrication of truly arbitrary microfluidic polydimethylsiloxane (PDMS) channels that are not achieved through typical soft lithography. The molds are printed directly from computer-aided design files, followed by simple dissolution using a solvent after molding PDMS, making rapid prototyping of microfluidic devices possible in hours. As part of the fabrication method, the solubility of several build materials in solvents and their effect on PDMS were investigated to remove the 3D-printed molds from inside the replicated PDMS microfluidic channels without damage. Technology limits, including surface roughness and resolution by comparing the designed channels with fabricated cylindrical channels with various diameters, are also characterized. We reproduced a 3D image of an actual human cerebral artery as cerebral artery-shaped PDMS channels with a diameter of 240 µm to prove the developed fabrication technique. It was confirmed that the fabricated vascular channels were free from any leakage by observing the fluorescence fluid fill.

A Field-Portable Cell Analyzer without a Microscope and Reagents.

This paper demonstrates a commercial-level field-portable lens-free cell analyzer called the NaviCell (No-stain and Automated Versatile Innovative cell analyzer) capable of automatically analyzing cell count and viability without employing an optical microscope and reagents. Based on the lens-free shadow imaging technique, the NaviCell (162 × 135 × 138 mm³ and 1.02 kg) has the advantage of providing analysis results with improved standard deviation between measurement results, owing to its large field of view. Importantly, the cell counting and viability testing can be analyzed without the use of any reagent, thereby simplifying the measurement procedure and reducing potential errors during sample preparation. In this study, the performance of the NaviCell for cell counting and viability testing was demonstrated using 13 and six cell lines, respectively. Based on the results of the hemocytometer (de facto standard), the error rate (ER) and coefficient of variation (CV) of the NaviCell are approximately 3.27 and 2.16 times better than the commercial cell counter, respectively. The cell viability testing of the NaviCell also showed an ER and CV performance improvement of 5.09 and 1.8 times, respectively, demonstrating sufficient potential in the field of cell analysis.

Non-planar PDMS microfluidic channels and actuators: a review.

This review examines the state of the art for manufacturing non-planar miniature channels and actuators from PDMS, where non-planar structures are defined here as those beyond simple extrusions of 2D designs, either with rounded or variable cross sections or with an emergence of the channel trajectory out-of-plane. The motivation for 3D PDMS structures and advances in their fabrication are described, focusing on geometries that were previously unachievable through conventional microfabrication. The motivation for non-planar microfluidic channels and actuators is first discussed and the existing literature is grouped into general fabrication themes and described. The structures are organized by their method of fabrication and evaluated based on their relevant properties, including the capability of producing structures with complex geometry, automation of the fabrication process, and minimum feature size. Additional properties are included for work in the more recently emerging field of non-planar PDMS actuators, where the feature size, actuation stroke, and actuation method are the key parameters of interest. In particular, this review considers the impact from recent advances in additive manufacturing, which now allow creation of truly arbitrary 3D structures down to ∼100 µm size scales.

Baicalein protects rat insulinoma INS-1 cells from palmitate-induced lipotoxicity by inducing HO-1.

OBJECTIVE: ß-Cell dysfunction plays a central role in the pathogenesis of type 2 diabetes (T2D), and the identification of novel approaches to improve ß-cell function is essential to treat this disease. Baicalein, a flavonoid originally isolated from the root of Scutellaria Baicalensis, has been shown to have beneficial effects on ß-cell function. Here, the authors investigated the molecular mechanism responsible for the protective effects of baicalein against palmitate (PA)-induced impaired ß-cell function, and placed focus on the role of heme oxygenase (HO)-1. METHODS: Rat pancreatic ß-cell line INS-1 cells or mouse pancreatic islets were cultured with PA (500 µM) to induce lipotoxicity in the presence or absence of baicalein (50 µM), and the expressions of the ER stress markers, ATF-3, CHOP and GRP78 were detected by Western blotting and/or qPCR. The involvement of HO-1 was evaluated by HO-1 siRNA transfection and using the HO-1 inhibitor ZnPP. RESULTS: Baicalein reduced PA-induced ER stress and inflammation and enhanced insulin secretion, and these effects were associated with the induction of HO-1. Furthermore, these protective effects were attenuated by ZnPP and by HO-1 siRNA. Pretreatment of PD98059 (an ERK inhibitor) significantly inhibited the protective effects of baicalein and blocked HO-1 induction. On the other hand, CO production by RuCO (a CO donor) ameliorated PA-induced ER stress, suggesting that CO production followed by HO-1 induction may contribute to the protective effects of baicalein against PA-induced ß-cell dysfunction. CONCLUSION: Baicalein protects pancreatic ß-cells from PA-induced ER stress and inflammation via an ERK-HO-1 dependent pathway. The authors suggest HO-1 induction in pancreatic ß-cells appears to be a promising therapeutic strategy for T2D.

Dielectrophoresis-assembled zeolitic imidazolate framework nanoparticle-coupled resonators for highly sensitive and selective gas detection.

This work reports on zeolitic imidazolate framework (ZIF)-coupled microscale resonators for highly sensitive and selective gas detection. The combination of microscale resonators and nanoscale materials simultaneously permits the benefit of larger capture area for adsorption from the resonator and enhanced surface adsorption capacity from the nanoscale ZIF structure. Dielectrophoresis (DEP) was demonstrated as a novel method for directly assembling concentrated ZIF nanoparticles on targeted regions of silicon resonant sensors. As part of the dielectrophoretic assembly process, the first ever measurements of the Clausius-Mossotti factor for ZIFs were conducted to determine optimal conditions for DEP assembly. The first ever real-time adsorption measurements of ZIFs were also performed to investigate the possibility of inherent gas selectivity. The ZIF-coupled resonators demonstrated sensitivity improvement up to 150 times over a bare silicon resonator with identical dimensions, and real-time adsorption measurements of ZIFs revealed different adsorption time constants for IPA and CO2.