Dermal macrophages set pain sensitivity by modulating the amount of tissue NGF through an SNX25-Nrf2 pathway.

Cross-talk between peripheral neurons and immune cells is important in pain sensation. We identified Snx25 as a pain-modulating gene in a transgenic mouse line with reduced pain sensitivity. Conditional deletion of Snx25 in monocytes and macrophages, but not in peripheral sensory neurons, in mice (Snx25cKO mice) reduced pain responses in both normal and neuropathic conditions. Bone marrow transplantation using Snx25cKO and wild-type mice indicated that macrophages modulated pain sensitivity. Expression of sorting nexin (SNX)25 in dermal macrophages enhanced expression of the neurotrophic factor NGF through the inhibition of ubiquitin-mediated degradation of Nrf2, a transcription factor that activates transcription of Ngf. As such, dermal macrophages set the threshold for pain sensitivity through the production and secretion of NGF into the dermis, and they may cooperate with dorsal root ganglion macrophages in pain perception.

A Deep Learning-Based Assay for Programmed Death Ligand 1 Immunohistochemistry Scoring in Non-Small Cell Lung Carcinoma: Does it Help Pathologists Score?

Several studies have developed various artificial intelligence (AI) models for immunohistochemical analysis of programmed death ligand 1 (PD-L1) in patients with non-small cell lung carcinoma; however, none have focused on specific ways by which AI-assisted systems could help pathologists determine the tumor proportion score (TPS). In this study, we developed an AI model to calculate the TPS of the PD-L1 22C3 assay and evaluated whether and how this AI-assisted system could help pathologists determine the TPS and analyze how AI-assisted systems could affect pathologists' assessment accuracy. We assessed the 4 methods of the AI-assisted system: (1 and 2) pathologists first assessed and then referred to automated AI scoring results (1, positive tumor cell percentage; 2, positive tumor cell percentage and visualized overlay image) for final confirmation, and (3 and 4) pathologists referred to the automated AI scoring results (3, positive tumor cell percentage; 4, positive tumor cell percentage and visualized overlay image) while determining TPS. Mixed-model analysis was used to calculate the odds ratios (ORs) with 95% CI for AI-assisted TPS methods 1 to 4 compared with pathologists' scoring. For all 584 samples of the tissue microarray, the OR for AI-assisted TPS methods 1 to 4 was 0.94 to 1.07 and not statistically significant. Of them, we found 332 discordant cases, on which the pathologists' judgments were inconsistent; the ORs for AI-assisted TPS methods 1, 2, 3, and 4 were 1.28 (1.06-1.54; P = .012), 1.29 (1.06-1.55; P = .010), 1.28 (1.06-1.54; P = .012), and 1.29 (1.06-1.55; P = .010), respectively, which were statistically significant. For discordant cases, the OR for each AI-assisted TPS method compared with the others was 0.99 to 1.01 and not statistically significant. This study emphasized the usefulness of the AI-assisted system for cases in which pathologists had difficulty determining the PD-L1 TPS.

Large-scale electron microscopic volume imaging of interfascicular oligodendrocytes in the mouse corpus callosum.

Single oligodendrocytes produce myelin sheaths around multiple axons in the central nervous system. Interfascicular oligodendrocytes (IOs) facilitate nerve conduction, but their detailed morphologies remain largely unknown. In the present study, we three-dimensionally reconstructed IOs in the corpus callosum of adult mouse using serial block face scanning electron microscopy. The cell bodies of IOs were morphologically polarized and extended thick processes from the cytoplasm-rich part of the cell. Processes originating from the cell body of each IO can be classified into two types: one myelinates an axon without branching, while the other type branches and each branch myelinates a distinct axon. Myelin sheaths originating from a particular IO have biased thicknesses, wrapping axons of a limited range of diameters. Consistent with this finding, IOs transduced and visualized with a rabies viral vector expressing GFP showed statistically significant variation in their myelination patterns. We further reconstructed the sheath immediately adjacent to that derived from each of the analyzed IOs; the thicknesses of the pair of sheaths were significantly correlated despite emanating from different IOs. These results suggest that a single axon could regulate myelin sheath thicknesses, even if the sheaths are derived from distinct IOs. Collectively, our results indicate that the IOs have their own myelin profiles defined by myelin thickness and axonal diameter although axons may regulate thickness of myelin sheath.

Detection of Simulated Fukushima Daichii Fuel Debris Using a Remotely Operated Vehicle at the Naraha Test Facility.

The use of robotics in harsh environments, such as nuclear decommissioning, has increased in recent years. Environments such as the Fukushima Daiichi accident site from 2011 and the Sellafield legacy ponds highlight the need for robotic systems capable of deployment in hazardous environments unsafe for human workers. To characterise these environments, it is important to develop robust and accurate localization systems that can be combined with mapping techniques to create 3D reconstructions of the unknown environment. This paper describes the development and experimental verification of a localization system for an underwater robot, which enabled the collection of sonar data to create 3D images of submerged simulated fuel debris. The system was demonstrated at the Naraha test facility, Fukushima prefecture, Japan. Using a camera with a bird's-eye view of the simulated primary containment vessel, the 3D position and attitude of the robot was obtained using coloured LED markers (active markers) on the robot, landmarks on the test-rig (passive markers), and a depth sensor on the robot. The successful reconstruction of a 3D image has been created through use of a robot operating system (ROS) node in real-time.

Coupling of the fusion and budding of giant phospholipid vesicles containing macromolecules.

Mechanisms that enabled primitive cell membranes to self-reproduce have been discussed based on the physicochemical properties of fatty acids; however, there must be a transition to modern cell membranes composed of phospholipids [Budin I, Szostak JW (2011) Proc Natl Acad Sci USA 108:5249-5254]. Thus, a growth-division mechanism of membranes that does not depend on the chemical nature of amphiphilic molecules must have existed. Here, we show that giant unilamellar vesicles composed of phospholipids can undergo the coupled process of fusion and budding transformation, which mimics cell growth and division. After gaining excess membrane by electrofusion, giant vesicles spontaneously transform into the budded shape only when they contain macromolecules (polymers) inside their aqueous core. This process is a result of the vesicle maximizing the translational entropy of the encapsulated polymers (depletion volume effect). Because the cell is a lipid membrane bag containing highly concentrated biopolymers, this coupling process that is induced by physical and nonspecific interactions may have a general importance in the self-reproduction of the early cellular compartments.

Formation of Epoxy Fatty Acids in Triacylglycerol Standards during Heating.

Epoxy fatty acid formation during heating was estimated using triolein (OOO) and trilinolein (LLL). Epoxy octadecanoic acids were found in heated OOO, while epoxy octadecenoic acids were found in heated LLL. The content of epoxy fatty acids increased with heating time, and trans-epoxy fatty acids were formed significantly more than cis-epoxy fatty acids. A comparison between OOO and LLL indicated that epoxy fatty acid formation was higher in the OOO than that in the LLL. Heating tests in the presence of α- tocopherol suggested that the formation of epoxy fatty acids could be suppressed by antioxidants.

Fully Transparent, Ultrathin Flexible Organic Electrochemical Transistors with Additive Integration for Bioelectronic Applications.

Optical transparency is highly desirable in bioelectronic sensors because it enables multimodal optical assessment during electronic sensing. Ultrathin (<5 µm) organic electrochemical transistors (OECTs) can be potentially used as a highly efficient bioelectronic transducer because they demonstrate high transconductance during low-voltage operation and close conformability to biological tissues. However, the fabrication of fully transparent ultrathin OECTs remains a challenge owing to the harsh etching processes of nanomaterials. In this study, fully transparent, ultrathin, and flexible OECTs are developed using additive integration processes of selective-wetting deposition and thermally bonded lamination. These processes are compatible with Ag nanowire electrodes and conducting polymer channels and realize unprecedented flexible OECTs with high visible transmittance (>90%) and high transconductance (≈1 mS) in low-voltage operations (<0.6 V). Further, electroencephalogram acquisition and nitrate ion sensing are demonstrated in addition to the compatibility of simultaneous assessments of optical blood flowmetry when the transparent OECTs are worn, owing to the transparency. These feasibility demonstrations show promise in contributing to human stress monitoring in bioelectronics.

Cell-free protein synthesis inside giant unilamellar vesicles analyzed by flow cytometry.

Lipid vesicles have been used as model cell systems, in which an in-vitro transcription-translation system (IVTT) is encapsulated to carry out intravesicular protein synthesis. Despite a large number of previous studies, a quantitative understanding of how protein synthesis inside the vesicles is affected by the lipid membrane remains elusive. This is mainly because of the heterogeneity in structural properties of the lipid vesicles used in the experiments. We investigated the effects of the phospholipid membrane on green fluorescent protein (GFP) synthesis occurring inside cell-sized giant unilamellar vesicles (GUV), which have a defined quantity of lipids relative to the reaction volume. We first developed a method to distinguish GUV from multilamellar vesicles using flow cytometry (FCM). Using this method, we investigated the time course of GFP synthesis using one of the IVTT, the PURE system, and found that phospholipid in the form of GUV has little effect on GFP synthesis based on three lines of investigation. (1) GFP synthesis inside the GUV was not dependent on the size of GUV (2) or on the fraction of cholesterol or anionic phospholipid constituting the GUV, and (3) GFP synthesis proceeded similarly in GUV and in the test tube. The present results suggest that GUV provides an ideal reaction environment that does not affect the internal biochemical reaction. On the other hand, we also found that internal GFP synthesis is strongly dependent on the chemical composition of the outer solution.

Effective pseudo-labeling based on heatmap for unsupervised domain adaptation in cell detection.

Cell detection is an important task in biomedical research. Recently, deep learning methods have made it possible to improve the performance of cell detection. However, a detection network trained with training data under a specific condition (source domain) may not work well on data under other conditions (target domains), which is called the domain shift problem. In particular, cells are cultured under different conditions depending on the purpose of the research. Characteristics, e.g., the shapes and density of the cells, change depending on the conditions, and such changes may cause domain shift problems. Here, we propose an unsupervised domain adaptation method for cell detection using a pseudo-cell-position heatmap, where the cell centroid is at the peak of a Gaussian distribution in the map and selective pseudo-labeling. In the prediction result for the target domain, even if the peak location is correct, the signal distribution around the peak often has a non-Gaussian shape. The pseudo-cell-position heatmap is thus re-generated using the peak positions in the predicted heatmap to have a clear Gaussian shape. Our method selects confident pseudo-cell-position heatmaps based on uncertainty and curriculum learning. We conducted numerous experiments showing that, compared with the existing methods, our method improved detection performance under different conditions.

New 29-nor-cycloartanes with a 3,4-seco- and a novel 2,3-seco-structure from the leaves of Sinocalycanthus chinensis.

Eight new triterpenoids, including sinocalycanchinensins A-E (1-5) with a 3,4-seco-29-nor-cycloartane skeleton, sinocalycanchinensin F (6) possessing a novel 2,3-seco-29-nor-cycloartane skeleton, and 29-nor-cycloartanes, sinocalycanchinensins G and H (7 and 8), have been isolated from the leaves of Sinocalycanthus chinensis. Their structures were elucidated on the basis of spectroscopic examinations. The cytotoxicities of the isolated new triterpenes against a panel of human cancer cell lines, including multi-drug resistant (MDR) cancer cell lines, were also evaluated. Compound 5 demonstrated enhanced cytotoxicity against MDR KB cells in the presence of colchicine, although all the compounds showed moderate or no cytotoxicities.

SNX25 regulates proinflammatory cytokine expression via the NF-κB signal in macrophages.

Innate immunity is the first line of defense against bacterial infection and is initiated by macrophages. Sorting nexin 25 (SNX25) is an SNX family member and is reported to negatively regulate TGF-ß signaling by enhancing TGF receptor degradation. However, few studies have focused on the relationship between SNX25 and the immune system. We knocked down SNX25 expression in macrophages and examined inflammatory cytokine expression, a hallmark of innate immunity, after lipopolysaccharide stimulation. SNX25 knockdown increased proinflammatory cytokine expression in RAW 264.7 cells. In addition, SNX25 knockdown activated the NF-κB signal by promoting ubiquitination of IκBα. These results suggest that SNX25 inhibits the NF-κB signal and thereby regulates proinflammatory cytokine expression in macrophages.

Weakly supervised cell instance segmentation under various conditions.

Cell instance segmentation is important in biomedical research. For living cell analysis, microscopy images are captured under various conditions (e.g., the type of microscopy and type of cell). Deep-learning-based methods can be used to perform instance segmentation if sufficient annotations of individual cell boundaries are prepared as training data. Generally, annotations are required for each condition, which is very time-consuming and labor-intensive. To reduce the annotation cost, we propose a weakly supervised cell instance segmentation method that can segment individual cell regions under various conditions by only using rough cell centroid positions as training data. This method dramatically reduces the annotation cost compared with the standard annotation method of supervised segmentation. We demonstrated the efficacy of our method on various cell images; it outperformed several of the conventional weakly-supervised methods on average. In addition, we demonstrated that our method can perform instance cell segmentation without any manual annotation by using pairs of phase contrast and fluorescence images in which cell nuclei are stained as training data.

Detection of association and fusion of giant vesicles using a fluorescence-activated cell sorter.

We have developed a method to evaluate the fusion process of giant vesicles using a fluorescence-activated cell sorter (FACS). Three fluorescent markers and FACS technology were used to evaluate the extent of association and fusion of giant vesicles. Two fluorescent markers encapsulated in different vesicle populations were used as association markers; when these vesicles associate, the two independent markers should be observed simultaneously in a single detection event. The quenched fluorescent marker and the dequencher, which were encapsulated in separate vesicle populations, were used as the fusion marker. When the internal aqueous solutions mix, the quenched marker is liberated by the dequencher and emits the third fluorescent signal. Although populations of pure POPC vesicles showed no detectable association or fusion, the same populations, oppositely charged by the exogenous addition of charged amphiphiles, showed up to 50% association and 30% fusion upon population analysis of 100,000 giant vesicles. Although a substantial fraction of the vesicles associated in response to a small amount of the charged amphiphiles (5% mole fraction compared to POPC alone), a larger amount of the charged amphiphiles (25%) was needed to induce vesicle fusion. The present methodology also revealed that the association and fusion of giant vesicles was dependent on size, with larger giant vesicles associating and fusing more frequently.

Spatial-Temporal Mitosis Detection in Phase-Contrast Microscopy via Likelihood Map Estimation by 3DCNN.

Automated mitotic detection in time-lapse phase-contrast microscopy provides us much information for cell behavior analysis, and thus several mitosis detection methods have been proposed. However, these methods still have two problems; 1) they cannot detect multiple mitosis events when there are closely placed. 2) they do not consider the annotation gaps, which may occur since the appearances of mitosis cells are very similar before and after the annotated frame. In this paper, we propose a novel mitosis detection method that can detect multiple mitosis events in a candidate sequence and mitigate the human annotation gap via estimating spatial-temporal likelihood map by 3DCNN. In this training, the loss gradually decreases with the gap size between ground-truth and estimation. This mitigates the annotation gaps. Our method outperformed the compared methods in terms of F1-score using challenging dataset that contains the data under four different conditions. Code is publicly available in https://github.com/naivete5656/MDMLM.

Stochasticity in gene expression in a cell-sized compartment.

The gene expression in a clonal cell population fluctuates significantly, and its relevance to various cellular functions is under intensive debate. A fundamental question is whether the fluctuation is a consequence of the complexity and redundancy in living cells or an inevitable attribute of the minute microreactor nature of cells. To answer this question, we constructed an artificial cell, which consists of only necessary components for the gene expression (in vitro transcription and translation system) and its boundary as a microreactor (cell-sized lipid vesicle), and investigated the gene expression noise. The variation in the expression of two fluorescent proteins was decomposed into the components that were correlated and uncorrelated between the two proteins using a method similar to the one used by Elowitz and co-workers to analyze the expression noise in E. coli. The observed fluctuation was compared with a theoretical model that expresses the amplitude of noise as a function of the average number of intermediate molecules and products. With the assumption that the transcripts are partly active, the theoretical model was able to well describe the noise in the artificial system. Furthermore, the same measurement for E. coli cells harboring an identical plasmid revealed that the E. coli exhibited a similar level of expression noise. Our results demonstrated that the level of fluctuation found in bacterial cells is mostly an intrinsic property that arises even in a primitive form of the cell.

Size control of giant unilamellar vesicles prepared from inverted emulsion droplets.

The production of giant lipid vesicles with controlled size and structure will be an important technology in the design of quantitative biological assays in cell-mimetic microcompartments. For establishing size control of giant vesicles, we investigated the vesicle formation process, in which inverted emulsion droplets are transformed into giant unilamellar vesicles (GUVs) when they pass through an oil/water interface. The relationship between the size of the template emulsion and the converted GUVs was studied using inverted emulsion droplets with a narrow size distribution, which were prepared by microfluidics. We successfully found an appropriate centrifugal acceleration condition to obtain GUVs that had a desired size and narrow-enough size distribution with an improved yield so that emulsion droplets can become the template for GUVs.

Identification and characterization of glycosyltransferases involved in the biosynthesis of soyasaponin I in Glycine max.

Triterpene saponins are a diverse group of compounds with a structure consisting of a triterpene aglycone and sugars. Identification of the sugar-transferase involved in triterpene saponin biosynthesis is difficult due to the structural complexity of triterpene saponin. Two glycosyltransferases from Glycine max, designated as GmSGT2 and GmSGT3, were identified and characterized. In vitro analysis revealed that GmSGT2 transfers a galactosyl group from UDP-galactose to soyasapogenol B monoglucuronide, and that GmSGT3 transfers a rhamnosyl group from UDP-rhamnose to soyasaponin III. These results suggest that soyasaponin I is biosynthesized from soyasapogenol B by successive sugar transfer reactions.

Population analysis of structural properties of giant liposomes by flow cytometry.

We used fluorescence flow cytometry to analyze the structural properties of populations of giant liposomes formed by different preparation methods. The inner aqueous volumes and nominal membrane surface areas of a large number of individual liposomes were measured simultaneously by using fluorescent markers. We compared these properties of liposomes prepared by the natural swelling method, the freeze-dried empty liposomes method, and the water-in-oil (W/O) emulsion method. A two-dimensional contour distribution map of the inner volume and the nominal surface area was used to elucidate the structural properties of liposomes over a wide range of liposome sizes. Lamellarity of liposomes was evaluated as the ratio of the nominal surface area to the theoretical surface area calculated from the liposome inner volume. This population analysis revealed the dependency of lamellarity on liposome volume: while the nominal surface areas of populations of liposomes prepared by the natural swelling and the freeze-dried empty liposome methods were widely distributed, those prepared by the W/O emulsion method had a narrower distribution within small values. Furthermore, with the latter method, the nominal surface area varied in proportion to the two-thirds power of the inner volume ranging for several orders of magnitude, indicating the liposomes had a thin membrane, which was constant for the wide volume range. The results as well as the methodology presented here would be useful in designing giant liposomes with desired properties.

Steroidal glycosides from the underground parts of Solanum sodomaeum.

A new steroidal glycoside has been isolated from the underground parts of Solanum sodomaeum L., along with seven known steroidal glycosides. Their chemical structures were determined on the basis of spectroscopic data and chemical evidence, and the structure of one known pregnane type glycoside was corrected. In addition, their antiproliferative activity against human promyelocytic leukemia (HL-60) cells was investigated, and five compounds exhibited stronger activity than cisplatin.