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Abstract Energy dispersion of electrons is the most fundamental property of the solid state physics. In models of electrons on a lattice with strong geometric frustration, the band dispersion of electrons can disappear due to the quantum destructive interference of the wavefunction. This is called a flat band, and it is known to be the stage for the emergence of various fascinating physical properties. It is a challenging task to realize this flat band in a real material. In this study, we performed first-principles calculations on two compounds, Pb $$_2$$ 2 As $$_2$$ 2 O $$_7$$ 7 and Pb $$_2$$ 2 Sn $$_2$$ 2 O $$_7$$ 7 , which are candidates to have flat bands near the Fermi level. Both compounds have electronic states close to flat bands, but the band width is significantly larger than that of Pb $$_2$$ 2 Sb $$_2$$ 2 O $$_7$$ 7 shown in previous research. Nevertheless, the density of states at the Fermi level of Pb $$_2$$ 2 As $$_2$$ 2 O $$_7$$ 7 is large enough to cause the system to undergo a ferromagnetic transition. In the case of Pb $$_2$$ 2 Sn $$_2$$ 2 O $$_7$$ 7 , pseudo-gap behavior near the Fermi level was observed. These findings underscore the importance of investigating the influence of flat bands on electronic energy dispersion, providing a crucial step toward understanding the emergence and characteristics of flat bands in novel materials.

Abstract We propose a method for integrating confocal amplitude and phase images obtained through dual-comb microscopy (DCM). DCM combines the benefits of confocal laser microscopy and quantitative phase microscopy, offering high axial resolution and scan-less imaging. By leveraging the coherence between confocal amplitude and phase images within the same DCM system, we accurately determine the number of phase wrapping iterations, thereby eliminating ambiguity in phase wrapping. We demonstrate this approach using samples with micrometer-range optical thickness and nanometer-scale surface roughness. The results demonstrate an expanded axial range, spanning from micrometers to millimeters, while maintaining nanometer-level axial resolution. This integrated DCM imaging technique enables the simultaneous acquisition of confocal amplitude image and absolute phase image, thus enhancing its potential for wide-axial-dynamic-range imaging across various applications.

Abstract Manufacturing regenerative medicine requires continuous monitoring of pluripotent cell culture and quality assessment while eliminating cell destruction and contaminants. In this study, we employed a novel method to monitor the pluripotency of stem cells through image analysis, avoiding the traditionally used invasive procedures. This approach employs machine learning algorithms to analyze stem cell images to predict the expression of pluripotency markers, such as OCT4 and NANOG, without physically interacting with or harming cells. We cultured induced pluripotent stem cells under various conditions to induce different pluripotent states and imaged the cells using bright-field microscopy. Pluripotency states of induced pluripotent stem cells were assessed using invasive methods, including qPCR, immunostaining, flow cytometry, and RNA sequencing. Unsupervised and semi-supervised learning models were applied to evaluate the results and accurately predict the pluripotency of the cells using only image analysis. Our approach directly links images to invasive assessment results, making the analysis of cell labeling and annotation of cells in images by experts dispensable. This core achievement not only contributes for safer and more reliable stem cell research but also opens new avenues for real-time monitoring and quality control in regenerative medicine manufacturing. Our research fills an important gap in the field by providing a viable, noninvasive alternative to traditional invasive methods for assessing pluripotency. This innovation is expected to make a significant contribution to improving regenerative medicine manufacturing because it will enable a more detailed and feasible understanding of cellular status during the manufacturing process.

Summary: Background: Intestinal epithelial cells (IECs) serve as robust barriers against potentially hostile luminal antigens and commensal microbiota. Epithelial barrier dysfunction enhances intestinal permeability, leading to leaky gut syndrome (LGS) associated with autoimmune and chronic inflammatory disorders. However, a causal relationship between LGS and systemic disorders remains unclear. Ap1m2 encodes clathrin adaptor protein complex 1 subunit mu 2, which facilitates polarized protein trafficking toward the basolateral membrane and contributes to the establishment of epithelial barrier functions. Methods: We generated IEC-specific Ap1m2-deficient (Ap1m2ΔIEC) mice with low intestinal barrier integrity as an LSG model and examined the systemic impact. Findings: Ap1m2ΔIEC mice spontaneously developed IgA nephropathy (IgAN)-like features characterized by the deposition of IgA–IgG immune complexes and complement factors in the kidney glomeruli. Ap1m2 deficiency markedly enhanced aberrantly glycosylated IgA in the serum owing to downregulation and mis-sorting of polymeric immunoglobulin receptors in IECs. Furthermore, Ap1m2 deficiency caused intestinal dysbiosis by attenuating IL-22-STAT3 signaling. Intestinal dysbiosis contributed to the pathogenesis of IgAN because antibiotic treatment reduced aberrantly glycosylated IgA production and renal IgA deposition in Ap1m2ΔIEC mice. Interpretation: IEC barrier dysfunction and subsequent dysbiosis by AP-1B deficiency provoke IgA deposition in the mouse kidney. Our findings provide experimental evidence of a pathological link between LGS and IgAN. Funding: AMED, AMED-CREST, JSPS Grants-in-Aid for Scientific Research, JST CREST, Fuji Foundation for Protein Research, and Keio University Program for the Advancement of Next Generation Research Projects.

Background: Investigating the association between plasma-free amino acids (PFAAs) and hyperuricemia (HU) in dyslipidemia (DL) and dyslipidemia with hypertension (DH) is crucial, as it could provide valuable insights into the pathophysiology of these conditions and contribute to the development of targeted prevention and management strategies. Therefore, in this study, we aimed to elucidate the associations between PFAAs and HU in individuals with DL and DH. Methods: We quantified PFAAs and uric acid levels among Japanese healthy subjects (n = 1311; HU, n = 57), subjects with DL (n = 1483; HU, n = 219), and subjects with DH (n = 1159; HU, n = 237). Results: The concentrations of most PFAAs showed significant differences between subjects without and with HU across all groups (p < 0.05 to 0.001). Adjusted logistic regression analyses revealed that certain PFAAs were consistently positively or negatively associated with HU across all groups. Specifically, in the DL group, alanine, tryptophan, and tyrosine showed significant positive associations with HU, while in the DH group, citrulline and glutamate exhibited similar positive associations (p < 0.05 to 0.001). Conversely, threonine in the healthy group (p < 0.05) and glutamine in the DL group (p < 0.05) demonstrated significant inverse associations with HU. Conclusions: This study revealed a potential close relationship between alterations in PFAA profiles and HU in dyslipidemia, without and with hypertension. The findings warrant further research to elucidate the role of altered amino acid and uric acid levels as potential disease biomarkers and therapeutic targets.

Abstract Liver fibrosis is assessed mainly by conventional staining or second harmonic generation (SHG) microscopy, which can only provide collagen content in fibrotic area. We propose to use polarization-resolved SHG (PR-SHG) microscopy to quantify liver fibrosis in terms of collagen fiber orientation and crystallization. Liver samples obtained from autopsy cases with fibrosis stage of F0–F4 were evaluated with an SHG microscope, and 12 consecutive PR-SHG images were acquired while changing the polarization azimuth angle of the irradiated laser from 0° to 165° in 15° increments using polarizer. The fiber orientation angle (φ) and degree (ρ) of collagen were estimated from the images. The SHG-positive area increased as the fibrosis stage progressed, which was well consistent with Sirius Red staining. The value of φ was random regardless of fibrosis stage. The mean value of ρ (ρ-mean), which represents collagen fiber crystallinity, varied more as fibrosis progressed to stage F3, and converged to a significantly higher value in F4 than in other stages. Spatial dispersion of ρ (ρ-entropy) also showed increased variation in the stage F3 and decreased variation in the stage F4. It was shown that PR-SHG could provide new information on the properties of collagen fibers in human liver fibrosis.

Abstract Herein, we integrated angle-scanning surface plasmon resonance (SPR) and angle-fixed SPR as a hybrid angular-interrogation SPR to enhance the sensing performance. Galvanometer-mirror-based beam angle scanning achieves a 100-Hz acquisition rate of both the angular SPR reflectance spectrum and the angle-fixed SPR reflectance, whereas the use of near-infrared light enhances the refractive index (RI) sensitivity, range, and precision compared with visible light. Simultaneous measurement of the angular SPR reflectance spectrum and angle-fixed SPR reflectance boosts the RI change range, RI resolution, and RI accuracy to 10–1–10–6 RIU, 2.24 × 10−6 RIU, and 5.22 × 10−6 RIU, respectively. The proposed hybrid SPR is a powerful tool for wide-dynamic-range RI sensing with various applications.

Abstract Excessive hip flexion torque to prioritize leg swings in the elderly is likely to be a factor that reduces their propulsive force and gait stability, but the mechanism is not clear. To understand the mechanism, we investigated how propulsive force, hip flexion torque, and margin of stability (MoS) change when only the hip spring stiffness is increased without changing the walking speed in the simple walking model, and verified whether the relationship holds in human walking. The results showed that at walking speeds between 0.50 and 1.75 m/s, increasing hip spring stiffness increased hip flexion torque and decreased the propulsive force and MoS in both the model and human walking. Furthermore, it was found that the increase in hip flexion torque was explained by the increase in spring stiffness, and the decreases in the propulsive force and MoS were explained by the increase in step frequency associated with the increase in spring stiffness. Therefore, the increase in hip flexion torque likely decreased the propulsive force and MoS, and this mechanism was explained by the intervening hip spring stiffness. Our findings may help in the control design of walking assistance devices, and in improving our understanding of elderly walking strategies.

Abstract The phonon-drag effect is useful for improving the thermoelectric performance, especially the Seebeck coefficient. Therefore, the phonon and electron transport properties of Si single crystals at different carrier densities were investigated, and the relationship between these properties and the phonon-drag effect was clarified. Phonon transport properties were determined using nanoindentation and spot-periodic heating radiation thermometry. The electron transport properties were determined based on the electrical conductivity of Si. The diffusive Seebeck coefficient derived from the electron transport properties was in good agreement with previous reports. However, the value of the phonon-drag Seebeck coefficient derived from the phonon transport properties is very low. This phenomenon suggests that phonons with a normal mean free path (MFP) do not contribute to the increase in the Seebeck coefficient; however, phonons with a long MFP and low frequency increase the Seebeck coefficient via the phonon-drag effect. Moreover, the phonon-drag effect was sufficiently pronounced even at 300 K and in the heavily doped region. These features are key in designing thermoelectric materials with enhanced performance derived from the phonon-drag effect.

Abstract Commensal bacteria-derived metabolites are critical in regulating the host immune system. Although the impact of gut microbiota-derived hydrophilic metabolites, such as short-chain fatty acids, on immune cell functions and development has been well documented, the immunomodulatory effects of gut microbiota-derived lipids are still of interest. Here, we report that lipid extracts from the feces of specific-pathogen-free (SPF), but not germ-free (GF), mice showed regulatory T (Treg)-cell-inducing activity. We conducted RP-HPLC-based fractionation and liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based lipidome profiling and identified two bioactive lipids, 9,10-dihydroxy-12Z-octadecenoic acid (9,10-DiHOME) and all-trans retinoic acid (atRA), with Treg-inducing activity in vitro. The luminal abundance of 9,10-DiHOME in the large intestine was significantly decreased by dextran sulfate sodium (DSS)-induced colitis, indicating that 9,10-DiHOME may be a potential biomarker of colitis. These observations implied that commensal bacteria-derived lipophilic metabolites might contribute to Treg development in the large intestine.

Abstract Iodine-containing formulations have been widely used to treat iodine deficiency and as antiseptics. Lecithin-bound iodine (LBI) has been approved to treat allergic diseases in Japan; however, its underlying mechanism remains unknown. In this study, we show that LBI ameliorated disease symptoms in an ovalbumin (OVA)-induced allergic rhinitis mouse model. LBI suppressed OVA-specific IgE production by attenuating germinal center (GC) reaction in the draining lymph nodes. The antiallergic effect of LBI is most likely attributed to increased serum iodine levels but not thyroid hormone levels. In vitro treatment of activated B cells with potassium iodide induced ferroptosis by increasing intracellular reactive oxygen species (ROS) and ferrous iron in a concentration-dependent manner. Accordingly, LBI diets increased ROS levels in GC B cells of the draining lymph nodes. This study suggests that iodine directly promotes ferroptosis in activated B cells and attenuates GC reactions, leading to the alleviation of allergic symptoms.

Pulmonary mucormycosis is rare in kidney transplant recipients and has a high mortality rate. We report a case of pulmonary mucormycosis presenting as round pneumonia 1 year and 1 month after the transplant. The diagnosis was confirmed by a percutaneous lung biopsy. A complete resection of the lung mass, followed by intravenous liposomal amphotericin B therapy, saved the life of the patient. In conclusion, early and prompt diagnosis followed by complete resection of the lesion in pulmonary mucormycosis is lifesaving.

Abstract Electron systems with strong geometrical frustrations have flat bands, and their unusual band dispersions are expected to induce a wide variety of physical properties. However, for the emergence of such properties, the Fermi level must be pinned within the flat band. In this study, we performed first-principles calculations on pyrochlore oxide Pb $$_2$$ 2 Sb $$_2$$ 2 O $$_7$$ 7 and theoretically clarified that the self-doping mechanism induces pinning of the Fermi level in the flat band in this system. Therefore, a very high density of states is realized at the Fermi level, and the ferromagnetic state transforms into the ground state via a flat band mechanism, although the system does not contain any magnetic elements. This compound has the potential to serve as a new platform for projecting the properties of flat band systems in the real world.

Abstract Various liquid biopsy methods have been developed for the non-invasive and early detection of diseases. In particular, the detection of circulating tumor cells (CTCs) and cancer-associated fibroblasts (CAFs) in blood has been receiving a great deal of attention. We have been developing systems and materials to facilitate such liquid biopsies. In this study, we further developed glass filters (with various patterns of holes, pitches, and non-adhesive coating) that can capture CTCs, but not white blood cells. We optimized the glass filters to capture CTCs, and demonstrated that they could be used to detect CTCs from lung cancer patients. We also used the optimized glass filters for detecting CAFs. Additionally, we further developed a system for visualizing the captured cells on the glass filters. Finally, we demonstrated that we could directly culture the captured cells on the glass filters. Based on these results, our high-performance glass filters appear to be useful for capturing and culturing CTCs and CAFs for further examinations.

Abstract This study focused on human contact behavior with objects and discussed countermeasures during the COVID-19 pandemic across 15 location types. Reducing contact with objects and disinfecting items can be implemented at a relatively low cost. We created a protocol for organizing the objects, and 1260 subjects who went outside during a day between December 3–7, 2020 in Tokyo and Kanagawa, Japan were surveyed. The participants touched 7317 objects in total; the most common objects were doors, chairs, baskets, elevator equipment, and cash. One-way analysis of variance and Scheffé’s multiple comparison test showed that supermarkets had the lowest mean and median values despite having the highest number of users, contact objects, and object types. Conversely, the values for hotels were the highest, significantly higher than that for other places, excluding amusement parks, workplaces, and schools and universities. Furthermore, the long-tailed frequency distribution of the number of objects suggests that the objects touched by many individuals are limited; thus, it is important to determine the objects to be prioritized for disinfection at each location. The data and protocol could inform infection countermeasures that properly address the contact realities as they pertain to people’s behavior and objects.

Abstract Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2) regulates the activities of numerous membrane proteins, including diacylglycerol(DAG)-activated TRPC3/6/7 channels. Although PIP2 binding is known to support DAG-activated TRP channel activity, its binding site remains unknown. We screened for PIP2 binding sites within TRPC6 channels through extensive mutagenesis. Using voltage-sensitive phosphatase (DrVSP), we found that Arg437 and Lys442, located in the channel’s pre-S1 domain/shoulder, are crucial for interaction with PIP2. To gain structural insights, we conducted computer protein–ligand docking simulations with the pre-S1 domain/shoulder of TRPC6 channels. Further, the functional significance of PIP2 binding to the pre-S1 shoulder was assessed for receptor-operated channel functions, cross-reactivity to DAG activation, and the kinetic model simulation. These results revealed that basic residues in the pre-S1 domain/shoulder play a central role in the regulation of PIP2-dependent gating. In addition, neutralizing mutation of K771 in the distal TRP box reversed the effect of PIP2 depletion from inhibiting to potentiating channel activity. A similar effect was seen in TRPV1 channels, which suggests that TRPC6 possesses a common but robust polarity switch mediating the PIP2-dependent effect. Overall, these mutagenesis studies reveal functional and structural insights for how basic residues and channel segments in TRP channels are controlled through phosphoinositides recognition.

PurposeAngiopoietin (Ang) 2 is released from vascular endothelial cells by the stimulation of vascular endothelial growth factor (VEGF)A. Ang2 increases the expression of leukocyte adhesion molecules on endothelial cells via nuclear factor κB. The aim of this study was to evaluate the effects of Ang2 and VEGFA on ocular autoimmune inflammation.MethodsWe measured the concentrations of Ang2 and VEGFA in vitreous samples among patients with uveitis. Vitreous samples were collected from 16 patients with idiopathic uveitis (uveitis group) and 16 patients with non-inflammatory eye disease (control group). Experimental autoimmune uveoretinitis (EAU) was induced in B10.BR mice with a human interphotoreceptor retinoid-binding protein-derived peptide. The retinochoroidal tissues of the EAU mice were removed, and the mRNA levels of Ang2 and VEGFA were examined. EAU mice treated with anti-Ang2, anti-VEGFA, a combination of anti-Ang2 and anti-VEGFA, anti-Ang2/VEGFA bispecific, or IgG control antibodies were clinically and histopathologically evaluated.ResultsThe protein levels of Ang2 and VEGFA were significantly higher in the vitreous samples of patients with uveitis than in controls (PConclusionsBased on these results, VEGFA and Ang2 were shown to be upregulated locally in the eye of both uveitis patients and models of uveitis. Dual inhibition of Ang2 and VEGFA is suggested to be a new therapeutic strategy for uveitis.

Medium-chain triglycerides (MCTs), which consist of medium-chain fatty acids (MCFAs), are unique forms of dietary fat with various health benefits. G protein–coupled 84 (GPR84) acts as a receptor for MCFAs (especially C10:0 and C12:0); however, GPR84 is still considered an orphan receptor, and the nutritional signaling of endogenous and dietary MCFAs via GPR84 remains unclear. Here, we showed that endogenous MCFA-mediated GPR84 signaling protected hepatic functions from diet-induced lipotoxicity. Under high-fat diet (HFD) conditions, GPR84-deficient mice exhibited nonalcoholic steatohepatitis (NASH) and the progression of hepatic fibrosis but not steatosis. With markedly increased hepatic MCFA levels under HFD, GPR84 suppressed lipotoxicity-induced macrophage overactivation. Thus, GPR84 is an immunomodulating receptor that suppresses excessive dietary fat intake–induced toxicity by sensing increases in MCFAs. Additionally, administering MCTs, MCFAs (C10:0 or C12:0, but not C8:0), or GPR84 agonists effectively improved NASH in mouse models. Therefore, exogenous GPR84 stimulation is a potential strategy for treating NASH.

The promising therapeutic implications of nanoparticles have spurred their development for biomedical applications. An eco-friendly methodology synthesizes gold nanoparticles using Cordyceps militaris, an edible mushroom (Cord-Au-NPs), using a quality-by-design approach (central composite design). UV-visible spectroscopy analysis revealed an absorption peak at 540–550 nm, thus confirming the synthesis of gold nanoparticles. Cord-Au-NPs have a crystalline structure, as evidenced by the diffraction peaks. The zeta potential value of −19.42 mV signifies the stability of Cord-Au-NPs. XRD study shows gold facets and EDX analysis revealed a strong peak of spherical nanoparticles in the gold region with a mean particle size of 7.18 nm and a polydispersity index of 0.096. The obtained peaks are closely associated with phenolic groups, lipids, and proteins, as examined by FTIR, suggesting that they function as the reducing agent. Cord-Au-NPs exhibited dose-dependent antioxidant, antidiabetic, and antibacterial activity. The method is eco-friendly, nontoxic, less time-consuming, and does not use synthetic materials, leading to higher capabilities in biomedical applications.

Abstract Challenge and threat models predict that once individuals become engaged with performance, their evaluations and cardiovascular response determine further outcomes. Although the role of challenge and threat in predicting performance has been extensively tested, few studies have focused on task engagement. We aimed to investigate task engagement in performance at the psychological and physiological levels. We accounted for physiological task engagement by examining blunted cardiovascular reactivity, the third possible cardiovascular response to performance, in addition to the challenge/threat responses. We expected that low psychological task engagement would be related to blunted cardiovascular reactivity during the performance. Gamers (N = 241) completed five matches of the soccer video game FIFA 19. We recorded psychological task engagement, heart rate reactivity, and the difference between goals scored and conceded. Lower psychological task engagement was related to blunted heart rate reactivity during the performance. Furthermore, poorer performance in the previous game was related to increased task engagement in the subsequent match. The findings extend existing literature by providing initial evidence that blunted cardiovascular reactivity may serve as the index of low task engagement.

Abstract Liver cancer is the fourth leading cause of cancer-related death. Hepatocellular carcinoma (HCC) is a primary liver cancer that results from chronic hepatitis caused by multiple predisposing factors such as viral infection, alcohol consumption, and non-alcoholic fatty liver disease. Accumulating studies have indicated that dysfunction of the gut epithelial barrier and hepatic translocation of gut microbes may be implicated in the pathogenesis of HCC. However, the translocated bacteria in HCC patients remains unclear. Here, we characterised tumour-associated microbiota in patients with liver cancer and focused on HCC. We observed that the number of amplicon sequence variants in tumour-associated microbiota was significantly higher compared with that in non-tumour regions of the liver. The tumour-associated microbiota consisted of Bacteroidetes, Firmicutes, and Proteobacteria as the dominant phyla. We identified an unclassified genus that belonged to the Bacteroides, Romboutsia, uncultured bacterium of Lachnospiraceae as a signature taxon for primary liver cancer. Additionally, we identified Ruminococcus gnavus as a signature taxon for HCC patients infected with hepatitis B and/or hepatitis C viruses. This study suggests that tumour microbiota may contribute to the pathology of HCC.

Abstract The human AlkB homolog family (ALKBH) of proteins play a critical role in some types of cancer. However, the expression and function of the lysine demethylase ALKBH4 in cancer are poorly understood. Here, we examined the expression and function of ALKBH4 in non-small-cell lung cancer (NSCLC) and found that ALKBH4 was highly expressed in NSCLC, as compared to that in adjacent normal lung tissues. ALKBH4 knockdown significantly induced the downregulation of NSCLC cell proliferation via cell cycle arrest at the G1 phase of in vivo tumour growth. ALKBH4 knockdown downregulated E2F transcription factor 1 (E2F1) and its target gene expression in NSCLC cells. ALKBH4 and E2F1 expression was significantly correlated in NSCLC clinical specimens. Moreover, patients with high ALKBH4 expression showed a poor prognosis, suggesting that ALKBH4 plays a pivotal tumour-promoting role in NSCLC.

A wide range of causative organisms can cause acute pyelonephritis (APN). However, in recent times, these pathogens have increasingly become resistant to most of the antibiotics making treatment difficult. This was a prospective observational single-center study with a aim to study the microbiological spectrum, resistance patterns, and clinical outcome of patients with APN conducted in a private tertiary care hospital in India. All adult patients hospitalized in the department of nephrology at our institute with a diagnosis of APN from February 2016 to May 2017 were included. Patients

Among non-dialysis-dependent chronic kidney disease (ND-CKD) patients, a low hematopoietic response to erythropoiesis-stimulating agents (ESAs) is a predictor for poor renal and cardiovascular outcome. To assess the method for evaluating hyporesponsiveness to ESA in patients with ND-CKD, a multicenter, prospective, observational study of 1,980 adult patients with ND-CKD with renal anemia was conducted. Darbepoetin alfa (DA) and iron supplement administrations were provided according to the recommendation of the attached document and the guidelines of JSDT (Japanese Society of Dialysis and Transplantation). The primary outcomes were progression of renal dysfunction and major adverse cardiovascular events. ESA responsiveness was assessed using pre-defined candidate formulae. During the mean follow-up period of 96 weeks, renal and cardiovascular disease (CVD) events occurred in 683 (39.6%) and 174 (10.1%) of 1,724 patients, respectively. Among pre-set candidate formulae, the one expressed by dividing the dose of DA by Hb level at the 12-week DA treatment was statistically significant in predicting renal (hazard ratio [HR], 1.449; 95% confidence interval [CI], 1.231-1.705; P

The liver is the main organ that regulates lipid and glucose metabolism. Ectopic lipid accumulation in the liver impairs insulin sensitivity and glucose metabolism. Lipoprotein lipase (LPL), mainly expressed in the adipose tissue and muscle, is a key enzyme that regulates lipid metabolism via the hydrolysis of triglyceride in chylomicrons and very-low-density lipoproteins. Here, we aimed to investigate whether the suppression level of hepatic lipid accumulation via overexpression of LPL in mouse liver leads to improved metabolism. To overexpress LPL in the liver, we generated an LPL-expressing adenovirus (Ad) vector using an improved Ad vector that exhibited considerably lower hepatotoxicity (Ad-LPL). C57BL/6 mice were treated with Ad vectors and simultaneously fed a high-fat diet (HFD). Lipid droplet formation in the liver decreased in Ad-LPL-treated mice relative to that in control Ad vector-treated mice. Glucose tolerance and insulin resistance were remarkably improved in Ad-LPL-treated mice compared to those in control Ad vector-treated mice. The expression levels of fatty acid oxidation-related genes, such as peroxisome proliferator-activated receptor α, carnitine palmitoyltransferase 1, and acyl-CoA oxidase 1, were 1.7-2.0-fold higher in Ad-LPL-treated mouse livers than that in control Ad-vector-treated mouse livers. Furthermore, hepatic LPL overexpression partly maintained mitochondrial content in HFD-fed mice. These results indicate that LPL overexpression in the livers of HFD-fed mice attenuates the accumulation of lipid droplets in the liver and improves glucose metabolism. These findings may enable the development of new drugs to treat metabolic syndromes such as type 2 diabetes mellitus and non-alcoholic fatty liver disease.

Background: The dysbiosis of gut microbiota has been implicated in the pathogenesis of inflammatory bowel diseases; however, the underlying mechanisms have not yet been elucidated. Heavily glycosylated mucin establishes a first-line barrier against pathogens and serves as a niche for microbial growth. Methods: To elucidate relationships among dysbiosis, abnormal mucin utilisation, and microbial metabolic dysfunction, we analysed short-chain fatty acids (SCFAs) and mucin components in stool samples of 40 healthy subjects, 49 ulcerative colitis (UC) patients, and 44 Crohn's disease (CD) patients from Japan. Findings: Levels of n-butyrate were significantly lower in stools of both CD and UC patients than in stools of healthy subjects. Correlation analysis identified seven bacterial species positively correlated with n-butyrate levels; the major n-butyrate producer, Faecalibacterium prausnitzii, was particularly underrepresented in CD patients, but not in UC patients. In UC patients, there were inverse correlations between mucin O-glycan levels and the production of SCFAs, such as n-butyrate, suggesting that mucin O-glycans serve as an endogenous fermentation substrate for n-butyrate production. Indeed, mucin-fed rodents exhibited enhanced n-butyrate production, leading to the expansion of RORgt+Treg cells and IgA-producing cells in colonic lamina propria. Microbial utilisation of mucin-associated O-glycans was significantly reduced in n-butyrate-deficient UC patients. Interpretation: Mucin O-glycans facilitate symbiosynthesis of n-butyrate by gut microbiota. Abnormal mucin utilisation may lead to reduced n-butyrate production in UC patients. Fund: Japan Society for the Promotion of Science, Health Labour Sciences Research Grant, AMED-Crest, AMED, Yakult Foundation, Keio Gijuku Academic Development Funds, The Aashi Grass Foundation, and The Canon Foundation. Keywords: Microbiota, Butyrate, Mucin, Inflammatory bowel disease

Abstract Zinc (Zn) is an essential nutrient and its deficiency causes immunodeficiency and skin disorders. Various cells including mast cells release Zn-containing granules when activated; however, the biological role of the released Zn is currently unclear. Here we report our findings that Zn transporter ZnT2 is required for the release of Zn from mast cells. In addition, we found that Zn and mast cells induce IL-6 production from inflammatory cells such as skin fibroblasts and promote wound healing, a process that involves inflammation. Zn induces the production of a variety of pro-inflammatory cytokines including IL-6 through signaling pathways mediated by the Zn receptor GPR39. Consistent with these findings, wound healing was impaired in mice lacking IL-6 or GPR39. Thus, our results show that Zn and mast cells play a critical role in wound healing through activation of the GPR39/IL-6 signaling axis.

Although quiescent hepatic stellate cells (HSCs) have been suggested to regulate hepatic blood flow, there is no direct evidence that quiescent HSCs display contractile abilities. Here, we developed a new method to quantitatively measure the contraction of single isolated HSCs and evaluated whether endothelin-1 (ET-1) induced contraction of HSCs in a non-activated state. HSCs isolated from mice were seeded on collagen gel containing fluorescent beads. The beads around a single HSC were observed gravitating toward the cell upon contraction. By recording the movement of each bead by fluorescent microscopy, the real-time contraction of HSCs was quantitatively evaluated. ET-1 induced a slow contraction of non-activated HSCs, which was inhibited by the non-muscle myosin II inhibitor blebbistatin, the calmodulin inhibitor W-7, and the ETA receptor antagonist ambrisentan. ET-1-induced contraction was also largely reduced in Ca2+-free conditions, but sustained contraction still remained. The tonic contraction was further diminished by the Rho-kinase inhibitor H-1152. The mRNA expression of P/Q-type voltage-dependent Ca2+ channels (VDCC), as well as STIM and Orai, constituents of store-operated channels (SOCs), was observed in mouse non-activated HSCs. ET-1-induced contraction was not affected by amlodipine, a VDCC blocker, whereas it was partly reduced by Gd3+ and amiloride, non-selective cation channel blockers. However, neither YM-58483 nor SKF-96365, which inhibit SOCs, had any effects on the contraction. These results suggest that ET-1 leads to Ca2+-influx through cation channels other than SOCs and produces myosin II-mediated contraction of non-activated HSCs via ETA receptors, as well as via mechanisms involving Ca2+-calmodulin and Rho kinase.

Background: Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disorder with a high prevalence, especially in industrialized countries. Dysbiosis of the intestinal microbiota has been observed in RA patients. For instance, new-onset untreated RA (NORA) is associated with the underrepresentation of the Clostridium cluster XIVa, including Lachnospiraceae, which are major butyrate producers, although the pathological relevance has remained obscure. Follicular regulatory T (TFR) cells play critical regulatory roles in the pathogenesis of autoimmune diseases, including RA. Reduced number of circulating TFR cells has been associated with the elevation of autoantibodies and disease severity in RA. However, the contribution of commensal microbe-derived butyrate in controlling TFR cell differentiation remains unknown. Methods: We examined the contribution of microbe-derived butyrate in controlling autoimmune arthritis using collagen-induced arthritis (CIA) and SKG arthritis models. We phenotyped autoimmune responses in the gut-associated lymphoid tissues (GALT) in the colon and joint-draining lymph nodes in the CIA model. We developed an in vitro CXCR5+Bcl-6+Foxp3+ TFR (iTFR) cell culture system and examined whether butyrate promotes the differentiation of iTFR cells. Findings: Microbe-derived butyrate suppressed the development of autoimmune arthritis. The immunization of type II collagen (CII) caused hypertrophy of the GALT in the colon by amplifying the GC reaction prior to the onset of the CIA. Butyrate mitigated these pathological events by promoting TFR cell differentiation. Butyrate directly induced the differentiation of functional TFR cells in vitro by enhancing histone acetylation in TFR cell marker genes. This effect was attributed to histone deacetylase (HDAC) inhibition by butyrate, leading to histone hyperacetylation in the promoter region of the TFR-cell marker genes. The adoptive transfer of the butyrate-treated iTFR cells reduced CII-specific autoantibody production and thus ameliorated the symptoms of arthritis. Interpretation: Accordingly, microbiota-derived butyrate serves as an environmental cue to enhance TFR cells, which suppress autoantibody production in the systemic lymphoid tissue, eventually ameliorating RA. Our findings provide mechanistic insights into the link between the gut environment and RA risk. Funding: This work was supported by AMED-Crest (16gm1010004h0101, 17gm1010004h0102, 18gm1010004h0103, and 19gm1010004s0104 to KH), the Japan Society for the Promotion of Science (JP17KT0055, JP16H01369, and JP18H04680 to KH; JP17K15734 to DT), Keio University Special Grant-in-Aid for Innovative Collaborative Research Projects (KH), Keio Gijuku Fukuzawa Memorial Fund for the Advancement of Education and Research (DT), the SECOM Science and Technology Foundation (KH), the Cell Science Research Foundation (KH), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (DT), the Suzuken Memorial Foundation (KH and DT), the Takeda Science Foundation (KH and DT), The Science Research Promotion Fund, and The Promotion and Mutual Aid Corporation for Private Schools of Japan (KH).

Patients with high serum ferritin and low transferrin saturation (TSAT) levels could be considered as presenting with dysutilization of iron for erythropoiesis. However, the long-term safety of iron administration in these patients has not been well established. An observational multicenter study was performed over 3 years. In 805 patients undergoing maintenance hemodialysis (MHD), we defined dysutilization of iron for erythropoiesis in patients with lower TSAT (

Detecting molecular targets in specimens from patients with lung cancer is essential for targeted therapy. Recently, we developed a highly sensitive, rapid-detection device (an immuno-wall device) that utilizes photoreactive polyvinyl alcohol immobilized with antibodies against a target protein via a streptavidin-biotin interaction. To evaluate its performance, we assayed epidermal growth factor receptor (EGFR) mutations, such as E746_A750 deletion in exon 19 or L858R substitution in exon 21, both of which are common in non-small cell lung cancer and important predictors of the treatment efficacy of EGFR tyrosine kinase inhibitors. The results showed that in 20-min assays, the devices detected as few as 1% (E746_A750 deletion) and 0.1% (L858R substitution) of mutant cells. Subsequent evaluation of detection of the mutations in surgically resected lung cancer specimens from patients with or without EGFR mutations and previously diagnosed using commercially available, clinically approved genotyping assays revealed diagnostic sensitivities of the immuno-wall device for E746_A750 deletion and L858R substitution of 85.7% and 87.5%, respectively, with specificities of 100% for both mutations. These results suggest that the immuno-wall device represents a good candidate next-generation diagnostic tool, especially for screening of EGFR mutations.

Several biomolecules potentially serve as promoters or inhibitors of calcification in dialysis patients which include fetuin A, matrix gla protein, osteopontin, osteoprotegerin, etc. The primary aim was to compare the biomarkers of coronary artery calcification (CAC) and to study its role as predictors of CAC in hemodialysis (HD) patients. Of 126 patients undergoing chronic HD, 100 patients completed the study. Blood samples were drawn for serum creatinine, electrolytes, calcium, phosphorus, Vitamin D3, parathyroid hormone (PTH), lipid profile, high sensitivity C-reactive protein, ferritin, fetuin A, and fibroblast growth factor-23 (FGF-23). Non-contrast Computed Tomography scan of the coronary arteries was conducted on all participants. Participants who were positive for CAC (P group) were compared with those negative for CAC (N group) using two sample t-test. Multiple logistic regression analysis was conducted to determine the predictors of CAC. The prevalence of vascular calcification (VC) was 60% with higher prevalence seen in males (71%), older age group, patients with dialysis vintage >5 years (27%), and diabetic population (62%). Mean serum phosphorus was significantly higher (P

CD4+CD25+ regulatory T cells (Treg), an essential subset for preventing autoimmune diseases, is implicated as a negative regulator in anti-tumor immunity. We found that metformin (Met) reduced tumor-infiltrating Treg (Ti-Treg), particularly the terminally-differentiated CD103+KLRG1+ population, and also decreased effector molecules such as CTLA4 and IL-10. Met inhibits the differentiation of naïve CD4+ T cells into inducible Treg (iTreg) by reducing forkhead box P3 (Foxp3) protein, caused by mTORC1 activation that was determined by the elevation of phosphorylated S6 (pS6), a downstream molecule of mTORC1. Rapamycin and compound C, an inhibitor of AMP-activated protein kinase (AMPK) restored the iTreg generation, further indicating the involvement of mTORC1 and AMPK. The metabolic profile of iTreg, increased Glut1-expression, and reduced mitochondrial membrane-potential and ROS production of Ti-Treg aided in identifying enhanced glycolysis upon Met-treatment. The negative impact of Met on Ti-Treg may help generation of the sustained antitumor immunity.

Abstract Transforming growth factor-β (TGF-β) is a cytokine produced by many cell types and implicated in cell growth, differentiation, apoptosis, and inflammation. It stimulates store-operated calcium entry (SOCE) through the calcium release-activated calcium (CRAC) channel Orai1/Stim1 in endometrial Ishikawa cells. Bone cells generate fibroblast growth factor (FGF) 23, which inhibits renal phosphate reabsorption and 1,25(OH)2D3 formation in concert with its co-receptor Klotho. Moreover, Klotho and FGF23 counteract aging and age-related clinical conditions. FGF23 production is dependent on Orai1-mediated SOCE and inflammation. Here, we explored a putative role of TGF-β2 in FGF23 synthesis. To this end, UMR106 osteoblast-like cells were cultured, Fgf23 transcript levels determined by qRT-PCR, FGF23 protein measured by ELISA, and SOCE analyzed by fluorescence optics. UMR106 cells expressed TGF-β receptors 1 and 2. TGF-β2 enhanced SOCE and potently stimulated the production of FGF23, an effect significantly attenuated by SB431542, an inhibitor of the transforming growth factor-β (TGF-β) type I receptor activin receptor-like kinases ALK5, ALK4, and ALK7. Furthermore, the TGF-β2 effect on FGF23 production was blunted by SOCE inhibitor 2-APB. We conclude that TGF-β2 induces FGF23 production, an effect involving up-regulation of SOCE.

Abstract The limits of conventional light microscopy (“Abbe-Limit“) depend critically on the numerical aperture (NA) of the objective lens. Imaging at large working distances or a large field-of-view typically requires low NA objectives, thereby reducing the optical resolution to the multi micrometer range. Based on numerical simulations of the intensity field distribution, we present an illumination concept for a super-resolution microscope which allows a three dimensional (3D) optical resolution around 150 nm for working distances up to the centimeter regime. In principle, the system allows great flexibility, because the illumination concept can be used to approximate the point-spread-function of conventional microscope optics, with the additional benefit of a customizable pupil function. Compared with the Abbe-limit using an objective lens with such a large working distance, a volume resolution enhancement potential in the order of 104 is estimated.

Serine/threonine protein kinase C (PKC) is activated by diacylglycerol that is released from membrane lipids by phospholipase C in response to activation of G protein-coupled receptors or receptor tyrosine kinases. PKC isoforms are particularly relevant for proliferation and differentiation of cells including osteoblasts. Osteoblasts/osteocytes produce fibroblast growth factor 23 (FGF23), a hormone regulating renal phosphate and vitamin D handling. PKC activates NFκB, a transcription factor complex controlling FGF23 expression. Here, we analyzed the impact of PKC on FGF23 synthesis. Fgf23 expression was analyzed by qRT-PCR in UMR106 osteoblast-like cells and in IDG-SW3 osteocytes, and FGF23 protein was measured by ELISA. Phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA), a PKC activator, up-regulated FGF23 production. In contrast, PKC inhibitors calphostin C, Gö6976, sotrastaurin and ruboxistaurin suppressed FGF23 formation. NFκB inhibitor withaferin A abolished the stimulatory effect of PMA on Fgf23. PKC is a powerful regulator of FGF23 synthesis, an effect which is at least partly mediated by NFκB.

Choline transporter-like protein 1 (CTL1) is highly expressed in glioma cells, and inhibition of CTL1 function induces apoptotic cell death. Therefore, CTL1 is a potential target molecule for glioma therapy. Here, we investigated the therapeutic mechanism underlying the antitumor effects of Amb4269951, a recently discovered novel CTL1 inhibitor, in the human glioma cell line U251MG, and evaluated its in vivo effects in a mouse xenograft model. Amb4269951 inhibited choline uptake and cell viability and increased caspase-3/7 activity. CTL1-mediated choline uptake is associated with cell viability, and the functional inhibition of CTL1 by Amb4269951 may promote apoptotic cell death via ceramide-induced suppression of the expression of survivin, an apoptotic inhibitory factor. Finally, Amb4269951 demonstrated an antitumor effect in a mice xenograft model by significantly inhibiting tumor growth without any weight loss. Amb4269951 is the lead compound in the treatment of glioma and exhibits a novel therapeutic mechanism. These results may lead to the development of novel anticancer drugs targeting the choline transporter CTL1, which has a different mechanism of action than conventional anticancer drugs against gliomas.

We studied the dialysis practice-patterns with regard to various aspects of chronic kidney disease (CKD) stage 5D, like anemia, mineral bone disease, vaccination, hospitalization, hypertension and cost of therapy. Four hundred and sixty-four adult hemodialysis (HD) patients from various dialysis centers of Mumbai were included in the study. The mean age of the study patients was 47.2 years. Temporary dialysis catheters were the most common initial vascular access. Thirteen percent of prevalent HD patients were on temporary catheters; 33% of patients had history of failure of arterio-venous fistula. The most common cause of failure was access thrombosis. About 75% of the patients had hemoglobin