Rational Engineering of XCaMPs, a Multicolor GECI Suite for In Vivo Imaging of Complex Brain Circuit Dynamics.

To decipher dynamic brain information processing, current genetically encoded calcium indicators (GECIs) are limited in single action potential (AP) detection speed, combinatorial spectral compatibility, and two-photon imaging depth. To address this, here, we rationally engineered a next-generation quadricolor GECI suite, XCaMPs. Single AP detection was achieved within 3-10 ms of spike onset, enabling measurements of fast-spike trains in parvalbumin (PV)-positive interneurons in the barrel cortex in vivo and recording three distinct (two inhibitory and one excitatory) ensembles during pre-motion activity in freely moving mice. In vivo paired recording of pre- and postsynaptic firing revealed spatiotemporal constraints of dendritic inhibition in layer 1 in vivo, between axons of somatostatin (SST)-positive interneurons and apical tufts dendrites of excitatory pyramidal neurons. Finally, non-invasive, subcortical imaging using red XCaMP-R uncovered somatosensation-evoked persistent activity in hippocampal CA1 neurons. Thus, the XCaMPs offer a critical enhancement of solution space in studies of complex neuronal circuit dynamics. VIDEO ABSTRACT.

Hematopoietic-Derived Galectin-3 Causes Cellular and Systemic Insulin Resistance.

In obesity, macrophages and other immune cells accumulate in insulin target tissues, promoting a chronic inflammatory state and insulin resistance. Galectin-3 (Gal3), a lectin mainly secreted by macrophages, is elevated in both obese subjects and mice. Administration of Gal3 to mice causes insulin resistance and glucose intolerance, whereas inhibition of Gal3, through either genetic or pharmacologic loss of function, improved insulin sensitivity in obese mice. In vitro treatment with Gal3 directly enhanced macrophage chemotaxis, reduced insulin-stimulated glucose uptake in myocytes and 3T3-L1 adipocytes and impaired insulin-mediated suppression of glucose output in primary mouse hepatocytes. Importantly, we found that Gal3 can bind directly to the insulin receptor (IR) and inhibit downstream IR signaling. These observations elucidate a novel role for Gal3 in hepatocyte, adipocyte, and myocyte insulin resistance, suggesting that Gal3 can link inflammation to decreased insulin sensitivity. Inhibition of Gal3 could be a new approach to treat insulin resistance.

Cell-matrix interface regulates dormancy in human colon cancer stem cells.

Cancer relapse after chemotherapy remains a main cause of cancer-related death. Although the relapse is thought to result from the propagation of resident cancer stem cells1, a lack of experimental platforms that enable the prospective analysis of cancer stem cell dynamics with sufficient spatiotemporal resolution has hindered the testing of this hypothesis. Here we develop a live genetic lineage-tracing system that allows the longitudinal tracking of individual cells in xenotransplanted human colorectal cancer organoids, and identify LGR5+ cancer stem cells that exhibit a dormant behaviour in a chemo-naive state. Dormant LGR5+ cells are marked by the expression of p27, and intravital imaging provides direct evidence of the persistence of LGR5+p27+ cells during chemotherapy, followed by clonal expansion. Transcriptome analysis reveals that COL17A1-a cell-adhesion molecule that strengthens hemidesmosomes-is upregulated in dormant LGR5+p27+ cells. Organoids in which COL17A1 is knocked out lose the dormant LGR5+p27+ subpopulation and become sensitive to chemotherapy, which suggests that the cell-matrix interface has a role in the maintenance of dormancy. Chemotherapy disrupts COL17A1 and breaks the dormancy in LGR5+p27+ cells through FAK-YAP activation. Abrogation of YAP signalling prevents chemoresistant cells from exiting dormancy and delays the regrowth of tumours, highlighting the therapeutic potential of YAP inhibition in preventing cancer relapse. These results offer a viable therapeutic approach to overcome the refractoriness of human colorectal cancer to conventional chemotherapy.

The nightside cloud-top circulation of the atmosphere of Venus.

Although Venus is a terrestrial planet similar to Earth, its atmospheric circulation is much different and poorly characterized1. Winds at the cloud top have been measured predominantly on the dayside. Prominent poleward drifts have been observed with dayside cloud tracking and interpreted to be caused by thermal tides and a Hadley circulation2-4; however, the lack of nightside measurements over broad latitudes has prevented the unambiguous characterization of these components. Here we obtain cloud-tracked winds at all local times using thermal infrared images taken by the Venus orbiter Akatsuki, which is sensitive to an altitude of about 65 kilometres5. Prominent equatorward flows are found on the nightside, resulting in null meridional velocities when these are zonally averaged. The velocity structure of the thermal tides was determined without the influence of the Hadley circulation. The semidiurnal tide was found to have an amplitude large enough to contribute to the maintenance of the atmospheric superrotation. The weakness of the mean meridional flow at the cloud top implies that the poleward branch of the Hadley circulation exists above the cloud top and that the equatorward branch exists in the clouds. Our results should shed light on atmospheric superrotation in other celestial bodies.

Highly porous nature of a primitive asteroid revealed by thermal imaging.

Carbonaceous (C-type) asteroids1 are relics of the early Solar System that have preserved primitive materials since their formation approximately 4.6 billion years ago. They are probably analogues of carbonaceous chondrites2,3 and are essential for understanding planetary formation processes. However, their physical properties remain poorly known because carbonaceous chondrite meteoroids tend not to survive entry to Earth's atmosphere. Here we report on global one-rotation thermographic images of the C-type asteroid 162173 Ryugu, taken by the thermal infrared imager (TIR)4 onboard the spacecraft Hayabusa25, indicating that the asteroid's boulders and their surroundings have similar temperatures, with a derived thermal inertia of about 300 J m-2 s-0.5 K-1 (300 tiu). Contrary to predictions that the surface consists of regolith and dense boulders, this low thermal inertia suggests that the boulders are more porous than typical carbonaceous chondrites6 and that their surroundings are covered with porous fragments more than 10 centimetres in diameter. Close-up thermal images confirm the presence of such porous fragments and the flat diurnal temperature profiles suggest a strong surface roughness effect7,8. We also observed in the close-up thermal images boulders that are colder during the day, with thermal inertia exceeding 600 tiu, corresponding to dense boulders similar to typical carbonaceous chondrites6. These results constrain the formation history of Ryugu: the asteroid must be a rubble pile formed from impact fragments of a parent body with microporosity9 of approximately 30 to 50 per cent that experienced a low degree of consolidation. The dense boulders might have originated from the consolidated innermost region or they may have an exogenic origin. This high-porosity asteroid may link cosmic fluffy dust to dense celestial bodies10.

Reinforced antimyeloma therapy via dual-lymphoid activation mediated by a panel of antibodies armed with bridging-BiTE.

Immunotherapy using bispecific antibodies including bispecific T-cell engager (BiTE) has the potential to enhance the efficacy of treatment for relapsed/refractory multiple myeloma. However, myeloma may still recur after treatment because of downregulation of a target antigen and/or myeloma cell heterogeneity. To strengthen immunotherapy for myeloma while overcoming its characteristics, we have newly developed a BiTE-based modality, referred to as bridging-BiTE (B-BiTE). B-BiTE was able to bind to both a human immunoglobulin G-Fc domain and the CD3 molecule. Clinically available monoclonal antibodies (mAbs) were bound with B-BiTE before administration, and the mAb/B-BiTE complex induced antitumor T-cell responses successfully while preserving and supporting natural killer cell reactivity, resulting in enhanced antimyeloma effects via dual-lymphoid activation. In contrast, any unwanted off-target immune-cell reactivity mediated by mAb/B-BiTE complexes or B-BiTE itself appeared not to be observed in vitro and in vivo. Importantly, sequential immunotherapy using 2 different mAb/B-BiTE complexes appeared to circumvent myeloma cell antigen escape, and further augmented immune responses to myeloma relative to those induced by mAb/B-BiTE monotherapy or sequential therapy with 2 mAbs in the absence of B-BiTE. Therefore, this modality facilitates easy and prompt generation of a broad panel of bispecific antibodies that can induce deep and durable antitumor responses in the presence of clinically available mAbs, supporting further advancement of reinforced immunotherapy for multiple myeloma and other refractory hematologic malignancies.

GPR120 is an omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects.

Omega-3 fatty acids (omega-3 FAs), DHA and EPA, exert anti-inflammatory effects, but the mechanisms are poorly understood. Here, we show that the G protein-coupled receptor 120 (GPR120) functions as an omega-3 FA receptor/sensor. Stimulation of GPR120 with omega-3 FAs or a chemical agonist causes broad anti-inflammatory effects in monocytic RAW 264.7 cells and in primary intraperitoneal macrophages. All of these effects are abrogated by GPR120 knockdown. Since chronic macrophage-mediated tissue inflammation is a key mechanism for insulin resistance in obesity, we fed obese WT and GPR120 knockout mice a high-fat diet with or without omega-3 FA supplementation. The omega-3 FA treatment inhibited inflammation and enhanced systemic insulin sensitivity in WT mice, but was without effect in GPR120 knockout mice. In conclusion, GPR120 is a functional omega-3 FA receptor/sensor and mediates potent insulin sensitizing and antidiabetic effects in vivo by repressing macrophage-induced tissue inflammation.

Postprandial fatty acid-binding protein 4 is associated with muscle insulin resistance.

AIMS/HYPOTHESIS: Fatty acid-binding protein 4 (FABP4) has been reported to act as a hepatic insulin resistance factor. We previously reported that fasting FABP4 was correlated with insulin resistance measurements derived from the glucose clamp, and another study reported that postprandial FABP4 levels were decreased in healthy volunteers but were not reported (or known) in participants with type 2 diabetes. We have limited knowledge about the direct effect of FABP4 on muscle cells. We investigated the postprandial FABP4 levels in participants with type 2 diabetes, and the basic mechanism of muscle insulin resistance and FABP4. METHODS: We performed a meal tolerance test and hyperinsulinaemic-euglycaemic clamp in 22 participants with type 2 diabetes and 26 participants without diabetes. We measured fasting and postprandial serum FABP4. We cultured mouse C2C12 muscle cells, and investigated the effect of FABP4 on glucose uptake. We analysed insulin signalling by western blot and insulin binding assay. RESULTS: The postprandial FABP4 level in participants with type 2 diabetes was higher than that in participants without diabetes. Participants without diabetes had lower postprandial FABP4 than fasting except for one participant, whereas one-third of participants with type 2 diabetes had higher postprandial FABP4 than fasting. Postprandial FABP4 was correlated with the muscle insulin resistance M/I value from a glucose clamp in participants without diabetes (r=-0.42, p<0.05). The increase in FABP4 after a meal correlated with the muscle insulin resistance M/I value (r=-0.44, p<0.05) and the difference between fasting and postprandial glucagon in participants with type 2 diabetes (r=0.36, p<0.05). FABP4 alone appears to increase glucose uptake, and the combination of FABP4 and insulin decreases glucose uptake when compared with insulin alone. FABP4 inhibits insulin signalling of muscle cells through decreases in phosphorylation of insulin receptor substrate 1 and Akt. The physiological concentration of FABP4 did not inhibit insulin binding to muscle cells. CONCLUSIONS/INTERPRETATION: These results suggested that the postprandial FABP4 level is associated with insulin resistance, and FABP4 may suppress insulin signals.

Heterozygous mutations in the straitjacket region of the latency-associated peptide domain of TGFB2 cause Camurati-Engelmann disease type II.

Camurati-Engelmann disease (CED) is an autosomal dominant bone dysplasia characterized by progressive hyperostosis of the skull base and diaphyses of the long bones. CED is further divided into two subtypes, CED1 and CED2, according to the presence or absence of TGFB1 mutations, respectively. In this study, we used exome sequencing to investigate the genetic cause of CED2 in three pedigrees and identified two de novo heterozygous mutations in TGFB2 among the three patients. Both mutations were located in the region of the gene encoding the straitjacket subdomain of the latency-associated peptide (LAP) of pro-TGF-ß2. Structural simulations of the mutant LAPs suggested that the mutations could cause significant conformational changes and lead to a reduction in TGF-ß2 inactivation. An activity assay confirmed a significant increase in TGF-ß2/SMAD signaling. In vitro osteogenic differentiation experiment using iPS cells from one of the CED2 patients showed significantly enhanced ossification, suggesting that the pathogenic mechanism of CED2 is increased activation of TGF-ß2 by loss-of-function of the LAP. These results, in combination with the difference in hyperostosis patterns between CED1 and CED2, suggest distinct functions between TGFB1 and TGFB2 in human skeletal development and homeostasis.

Graded arc beam in light needle microscopy for axially resolved, rapid volumetric imaging without nonlinear processes.

High-speed three-dimensional (3D) imaging is essential for revealing the structure and functions of biological specimens. Confocal laser scanning microscopy has been widely employed for this purpose. However, it requires a time-consuming image-stacking procedure. As a solution, we previously developed light needle microscopy using a Bessel beam with a wavefront-engineered approach [Biomed. Opt. Express13, 1702 (2022)10.1364/BOE.449329]. However, this method applies only to multiphoton excitation microscopy because of the requirement to reduce the sidelobes of the Bessel beam. Here, we introduce a beam that produces a needle spot while eluding the intractable artifacts due to the sidelobes. This beam can be adopted even in one-photon excitation fluorescence 3D imaging. The proposed method can achieve real-time, rapid 3D observation of 200-nm particles in water at a rate of over 50 volumes per second. In addition, fine structures, such as the spines of neurons in fixed mouse brain tissue, can be visualized in 3D from a single raster scan of the needle spot. The proposed method can be applied to various modalities in biological imaging, enabling rapid 3D image acquisition.

Single-Cell Phenotyping of CD73 Expression Reveals the Diversity of the Tumor Immune Microenvironment and Reflects the Prognosis of Bladder Cancer.

The cutting edge of cancer immunotherapy extends to ecto-5'-nucleotidase (CD73), a cell membrane enzyme that targets the metabolism of extracellular adenosine. We herein focused on the expression of CD73 to clarify the state of CD73 positivity in cancer immunity and tumor microenvironment, thereby revealing a new survival predictor for patients with bladder cancer (BCa). We used clinical tissue microarrays of human BCa and simultaneously performed the fluorescent staining of cell type-specific markers (CD3, CD8, Foxp3, programmed cell death protein 1, and programmed death-ligand 1 [PD-L1]) and CD73 together with DAPI for nuclear staining. In total, 156 participants were included. Multiplexed cellular imaging revealed a unique interaction between CD73 expression and CD8+ cytotoxic T cells (CTLs) and Foxp3+ regulatory T (Treg) cells in human BCa, showing the high infiltration of CD8+CD73+ CTLs and Foxp3+CD73+ Treg cells in tumors to be associated with tumorigenesis and poor prognosis in BCa. Interestingly, from a biomarker perspective, the high infiltration of CD73+ Treg cells in tumors was identified as an independent risk factor for overall survival in addition to clinicopathologic features. Regarding the relationship between immune checkpoint molecules and CD73 expression, both CD73+ CTLs and CD73+ Treg cells tended to coexpress programmed cell death protein 1 as tumor invasiveness and nuclear grade increased. Additionally, they may occupy a spatial niche located distantly from PD-L1+ cells in tumors to interfere less with the cancerous effects of PD-L1+ cells. In conclusion, the present results on the status of CD73 in cancer immunity suggest that CD73 expression on specific T-cell types has a negative immunoregulatory function. These findings may provide further insights into the immunobiological landscape of BCa, which may be translationally linked to improvements in future immunotherapy practice.

Identification of Quiescent LGR5+ Stem Cells in the Human Colon.

BACKGROUND & AIMS: In the mouse intestinal epithelium, Lgr5+ stem cells are vulnerable to injury, owing to their predominantly cycling nature, and their progenies de-differentiate to replenish the stem cell pool. However, how human colonic stem cells behave in homeostasis and during regeneration remains unknown. METHODS: Transcriptional heterogeneity among colonic epithelial cells was analyzed by means of single-cell RNA sequencing analysis of human and mouse colonic epithelial cells. To trace the fate of human colonic stem or differentiated cells, we generated LGR5-tdTomato, LGR5-iCasase9-tdTomato, LGR5-split-Cre, and KRT20-ERCreER knock-in human colon organoids via genome engineering. p27+ dormant cells were further visualized with the p27-mVenus reporter. To analyze the dynamics of human colonic stem cells in vivo, we orthotopically xenotransplanted fluorescence-labeled human colon organoids into immune-deficient mice. The cell cycle dynamics in xenograft cells were evaluated using 5-ethynyl-2'-deoxyuridine pulse-chase analysis. The clonogenic capacity of slow-cycling human stem cells or differentiated cells was analyzed in the context of homeostasis, LGR5 ablation, and 5-fluorouracil-induced mucosal injury. RESULTS: Single-cell RNA sequencing analysis illuminated the presence of nondividing LGR5+ stem cells in the human colon. Visualization and lineage tracing of slow-cycling LGR5+p27+ cells and orthotopic xenotransplantation validated their homeostatic lineage-forming capability in vivo, which was augmented by 5-FU-induced mucosal damage. Transforming growth factor-ß signaling regulated the quiescent state of LGR5+ cells. Despite the plasticity of differentiated KRT20+ cells, they did not display clonal growth after 5-FU-induced injury, suggesting that occupation of the niche environment by LGR5+p27+ cells prevented neighboring differentiated cells from de-differentiating. CONCLUSIONS: Our results highlight the quiescent nature of human LGR5+ colonic stem cells and their contribution to post-injury regeneration.

Visualizing spatiotemporal dynamics of multicellular cell-cycle progression.

The cell-cycle transition from G1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G1 phase nuclei red and those in S/G2/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resolution to help us better understand how the cell cycle is coordinated with various biological events.

Epstein-Barr virus reactivation in peripheral B lymphocytes induces IgM-type thyrotropin receptor autoantibody production in patients with Graves' disease.

Epstein-Barr virus (EBV) is a human herpes virus that latently infects B lymphocytes. When EBV is reactivated, host B cells differentiate into plasma cells and produce IgM-dominant antibodies as well as many progeny virions. The aims of the present study were to confirm the IgM dominance of thyrotropin-receptor antibodies (TRAbs) produced by EBV reactivation and investigate the roles of TRAb-IgM in Graves' disease. Peripheral blood mononuclear cells (PBMCs) containing TRAb-producing cells were stimulated for EBV reactivation, and TRAb-IgM and TRAb-IgG were measured by ELISA. TRAb-IgM were purified and TSH-binding inhibitory activities were assessed using a radio-receptor assay. Porcine thyroid follicular epithelial cells were cultured with TRAb-IgM and/or complements to measure the intracellular levels of cAMP and the amount of LDH released. TRAb-IgM/TRAb-IgG (the MG ratio) was examined in sequential serum samples of Graves' disease and compared among groups of thyroid function. The results obtained showed that IgM-dominant TRAb production was induced by EBV reactivation. TRAb-IgM did not inhibit TSH binding to TSH receptors and did not transduce hormone-producing signals. However, it destroyed thyroid follicular epithelial cells with complements. The MG ratio was significantly higher in samples of hyperthyroidism or hypothyroidism than in those with normal function or in healthy controls. A close relationship was observed between TRAb-IgM produced by EBV reactivation and the development and exacerbation of Graves' disease. The present results provide novel insights for the development of prophylaxis and therapeutics for Graves' disease.

Extended Depth of Focus Two-Photon Light-Sheet Microscopy for In Vivo Fluorescence Imaging of Large Multicellular Organisms at Cellular Resolution.

Two-photon excitation in light-sheet microscopy advances applications to live imaging of multicellular organisms. In a previous study, we developed a two-photon Bessel beam light-sheet microscope with a nearly 1-mm field of view and less than 4-µm axial resolution, using a low magnification (10×), middle numerical aperture (NA 0.5) detection objective. In this study, we aimed to construct a light-sheet microscope with higher resolution imaging while maintaining the large field of view, using low magnification (16×) with a high NA 0.8 objective. To address potential illumination and detection mismatch, we investigated the use of a depth of focus (DOF) extension method. Specifically, we used a stair-step device composed of five-layer annular zones that extended DOF two-fold, enough to cover the light-sheet thickness. Resolution measurements using fluorescent beads showed that the reduction in resolutions was small. We then applied this system to in vivo imaging of medaka fish and found that image quality degradation at the distal site of the beam injection could be compensated. This demonstrates that the extended DOF system combined with wide-field two-photon light-sheet microscopy offers a simple and easy setup for live imaging application of large multicellular organism specimens with sub-cellular resolution.

Tumor-targeted fluorescence labeling systems for cancer diagnosis and treatment.

Conventional imaging techniques are available for clinical identification of tumor sites. However, detecting metastatic tumor cells that are spreading from primary tumor sites using conventional imaging techniques remains difficult. In contrast, fluorescence-based labeling systems are useful tools for detecting tumor cells at the single-cell level in cancer research. The ability to detect fluorescent-labeled tumor cells enables investigations of the biodistribution of tumor cells for the diagnosis and treatment of cancer. For example, the presence of fluorescent tumor cells in the peripheral blood of cancer patients is a predictive biomarker for early diagnosis of distant metastasis. The elimination of fluorescent tumor cells without damaging normal tissues is ideal for minimally invasive treatment of cancer. To capture fluorescent tumor cells within normal tissues, however, tumor-specific activated target molecules are needed. This review focuses on recent advances in tumor-targeted fluorescence labeling systems, in which indirect reporter labeling using tumor-specific promoters is applied to fluorescence labeling of tumor cells for the diagnosis and treatment of cancer. Telomerase promoter-dependent fluorescence labeling using replication-competent viral vectors produces fluorescent proteins that can be used to detect and eliminate telomerase-positive tumor cells. Tissue-specific promoter-dependent fluorescence labeling enables identification of specific tumor cells. Vimentin promoter-dependent fluorescence labeling is a useful tool for identifying tumor cells that undergo epithelial-mesenchymal transition (EMT). The evaluation of tumor cells undergoing EMT is important for accurately assessing metastatic potential. Thus, tumor-targeted fluorescence labeling systems represent novel platforms that enable the capture of tumor cells for the diagnosis and treatment of cancer.

Thrombin induces a temporal biphasic vascular response through the differential phosphorylation of endothelial nitric oxide synthase via protease-activated receptor-1 and protein kinase C.

Endothelial nitric oxide synthase (eNOS) is a critical regulatory enzyme that controls vascular tone via the production of nitric oxide. Although thrombin also modulates vascular tone predominantly via the activation of protease-activated receptors (PARs), the time course and mechanisms involved in how thrombin controls eNOS enzymatic activity are unknown. eNOS enzymatic activity is enhanced by the phosphorylation of eNOS-Ser1177 and reduced by the phosphorylation of eNOS-Thr495. In this study, we hypothesized that thrombin regulates vascular tone through the differential phosphorylation of eNOS. Using rat descending aorta, we show that thrombin modulates vascular tone in an eNOS-dependent manner via activated PAR-1. We also show that thrombin causes a temporal biphasic response. Protein kinase C (PKC) is associated with second phase of thrombin-induced response. Western blot analysis demonstrated thrombin phosphorylated eNOS-Ser1177 and eNOS-Thr495 in human umbilical vein endothelial cells. A PKC inhibitor suppressed the thrombin-induced phosphorylation of eNOS-Thr495, but not that of eNOS-Ser1177. Our results suggest that thrombin induces a temporal biphasic vascular response through the differential phosphorylation of eNOS via activated PAR-1. Thrombin causes transient vasorelaxation by the phosphorylation of eNOS-Ser1177, and subsequent attenuation of vasorelaxation by the phosphorylation of eNOS-Thr495 via PKC, leading to the modulation of vascular tone.

Computer-Aided Detection of Quantitative Signatures for Breast Fibroepithelial Tumors Using Label-Free Multi-Photon Imaging.

Fibroadenomas (FAs) and phyllodes tumors (PTs) are major benign breast tumors, pathologically classified as fibroepithelial tumors. Although the clinical management of PTs differs from FAs, distinction by core needle biopsy diagnoses is still challenging. Here, a combined technique of label-free imaging with multi-photon microscopy and artificial intelligence was applied to detect quantitative signatures that differentiate fibroepithelial lesions. Multi-photon excited autofluorescence and second harmonic generation (SHG) signals were detected in tissue sections. A pixel-wise semantic segmentation method using a deep learning framework was used to separate epithelial and stromal regions automatically. The epithelial to stromal area ratio and the collagen SHG signal strength were investigated for their ability to distinguish fibroepithelial lesions. An image segmentation analysis with a pixel-wise semantic segmentation framework using a deep convolutional neural network showed the accurate separation of epithelial and stromal regions. A further investigation, to determine if scoring the epithelial to stromal area ratio and the SHG signal strength within the stromal area could be a marker for differentiating fibroepithelial tumors, showed accurate classification. Therefore, molecular and morphological changes, detected through the assistance of computational and label-free multi-photon imaging techniques, enable us to propose quantitative signatures for epithelial and stromal alterations in breast tissues.

Multiplexed single-cell pathology reveals the association of CD8 T-cell heterogeneity with prognostic outcomes in renal cell carcinoma.

Despite the high sensitivity of renal cell carcinoma (RCC) to immunotherapy, RCC has been recognized as an unusual disease in which CD8+ T-cell infiltration into the tumor beds is related to a poor prognosis. To approach the inner landscape of immunobiology of RCC, we performed multiplexed seven-color immunohistochemistry (CD8, CD39, PD-1, Foxp3, PD-L1, and pan-cytokeratin AE1/AE3 with DAPI), which revealed the automated single-cell counts and calculations of individual cell-to-cell distances. In total, 186 subjects were included, in which CD39 was used as a marker for distinguishing tumor-specific (CD39+) and bystander (CD39-) T-cells. Our clear cell RCC cohort also revealed a poor prognosis if the tumor showed increased CD8+ T-cell infiltration. Intratumoral CD8+CD39+ T-cells as well as their exhausted CD8+CD39+PD-1+ T-cells in the central tumor areas enabled the subgrouping of patients according to malignancy. Analysis using specimens post-antiangiogenic treatment revealed a dramatic increase in proliferative Treg fraction Foxp3+PD-1+ cells, suggesting a potential mechanism of hyperprogressive disease after uses of anti-PD-1 antibody. Our cell-by-cell study platform provided spatial information on tumors, where bystander CD8+CD39- T-cells were dominant in the invasive margin areas. We uncovered a potential interaction between CD8+CD39+PD-1+ T-cells and Foxp3+PD-1+ Treg cells due to cell-to-cell proximity, forming a spatial niche more specialized in immunosuppression under PD-1 blockade. A paradigm shift to the immunosuppressive environment was more obvious in metastatic lesions; rather the infiltration of Foxp3+ and Foxp3+PD-1+ Treg cells was more pronounced. With this multiplexed single-cell pathology technique, we revealed further insight into the immunobiological standing of RCC.

Morphological divergence in the curvature of human femoral diaphyses: Tracing the central mass distributions of cross-sections.

The diaphysis of the human femoral bone has a physiological anterior curvature; additionally, there is a curvature to the medial side or lateral side. In addition to compression stress from gravity during standing, walking, and running, these bones are continuously exposed to complex stresses from the traction forces of the various strong muscles attached to them. The femoral diaphysis is subjected to these mechanical stresses, and the direction and size of its curvature are defined according to Wolff's law and the mechanostat theory of Frost. The purpose of this study was to quantitatively evaluate the curvature of the femoral diaphysis in Japanese skeletons by determining the curve connecting the central mass distributions (CMD) of cross-sectional images. A total of 90 right femora (46 males and 44 females) were randomly selected from modern Japanese skeletal specimens. Full-length images of these bones were acquired using a clinical computed tomography scanner. The range between the lower end of the lesser trochanter and the adductor tubercle of each femur was divided at regular intervals to obtain ten planes, and nine levels were analyzed. The CMD curve was determined by connecting the CMDs of each of the nine cross-sections. First, the CMD of a cross-section in each of the nine slices was calculated, and the nine trajectories were superimposed from above. Then, by converting the shape of the entire CMD curve to superimpose the coordinates of the endpoint on the starting point, a closed arc representing the curvature of the femur was determined. For both males and females, the patterns varied from mostly medial to largely lateral curvature. The size of the curvature also varied for individuals. By analyzing only the coordinates of the vertex of the CMD curve of each femoral bone, the outlines of the diaphyseal curvatures could be recognized. The femora were thereby divided into two groups: medial bending and lateral bending. Considering males and females together, the number in the lateral-curvature group (n = 51) was larger than that in the medial-curvature group (n = 39). Moreover, the average age of the lateral-curvature group was significantly higher than that of the medial-curvature group (p < 0.05). In males, with an increase in the cortical bone proportion of the cross-sectional area, the anterior vertex of diaphyseal bending tended to be more prominent. This cortical proportion was significantly higher in the medial-curvature groups than in the lateral-curvature group (p < 0.01). The phenomena observed in this study may be related to pathophysiologies such as atypical fractures of the femur and osteoarthritis of the knee joints.