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Abstract Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides that bind to RNAs through Watson–Crick base pairings. They are actively being developed as therapeutics for various human diseases. ASOs containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are known to trigger innate immune responses via interaction with toll-like receptor 9 (TLR9). However, the TLR9-stimulatory properties of ASOs, specifically those with lengths equal to or less than 20 nucleotides, phosphorothioate linkages, and the presence and arrangement of sugar-modified nucleotides—crucial elements for ASO therapeutics under development—have not been thoroughly investigated. In this study, we first established SY-ODN18, an 18-nucleotide phosphorothioate oligodeoxynucleotide with sufficient TLR9-stimulatory activity. We demonstrated that an unmethylated CpG motif near its 5′-end was indispensable for TLR9 activation. Moreover, by utilizing various sugar-modified nucleotides, we systematically generated model ASOs, including gapmer, mixmer, and fully modified designs, in accordance with the structures of ASO therapeutics. Our results illustrated that introducing sugar-modified nucleotides in such designs significantly reduces TLR9-stimulatory activity, even without methylation of CpG motifs. These findings would be useful for drug designs on several types of ASOs.

Muscle regeneration requires the contribution and communication of various different cell types. Here, Nawaz et al. show that CD206+ macrophages inhibit the secretion of the promyogenic factor follistatin by fibro-adipogenic progenitor cells, impeding myogenesis and muscle regeneration.

The elasticity of biological tissues is one of the physical characteristics of tissues and has attracted attention as a clinical diagnostic parameter. The elasticity can be determined on the microscopic scale with speed of sound (SoS) measurements using acoustic microscopy. In SoS measurements, a thin-sliced section is attached to a glass slide in the same manner as a light microscopic specimen. There are two main methods for preparing thin sections: paraffin-embedding and frozen-section. The frozen-section method requires fewer processing steps from sectioning to measurement and is considered to reduce artifacts in the sample compared with the paraffin-embedding method. Both methods need fixatives to keep tissue structures. Many reports of measurements using frozen sections are focused on soft tissues with relatively high protein contents. In this study, we determined the SoS in thin sections of four types of organs (brain, heart, liver, and kidney) prepared using two different methods (paraffin-embedding and frozen-section) and four different chemical fixatives (formalin, Karnovsky fixative (KF) 0.5% and 2.0% glutaraldehyde, and ethanol). The SoS in heart and liver samples prepared using KF showed good agreement with reported values for raw samples. For samples fixed with KF, the SoS increased as the glutaraldehyde concentration increased from 0.5% to 2.0%. A brain tumor sample was processed with KF 0.5%, and the SoS in the tumor was significantly higher than that in the non-tumor area. The results confirmed that it is possible to measure the SoS in brain samples with low protein contents using appropriate fixatives.

Background Electrical stimulation of the cerebellar fastigial nucleus (FN) reduces the infarct size induced by middle cerebral artery occlusion in rats. FN stimulation confers long‐lasting protection from brain injury; however, its underlying mechanism is not yet understood. We aimed to elucidate the mechanism by which FN stimulation exerts neuroprotection. We hypothesized that the neuroprotective effect of FN stimulation involves activation of cholinergic pathways, which increases reactive oxygen species (ROS) production by opening mitochondrial K+ATP channels, thus leading to an increase in UCP4 (uncoupling protein 4) expression and subsequent neuroprotection. Methods FN stimulation was performed for 1 hour in rats. The UCP4 protein and mRNA levels were measured by western blot, dot blot, and in situ hybridization. Carbachol was applied following UCP4‐promoter tdTomato reporter vector transfection of the rat primary cortical cell culture (in vitro) and rat brain (in vivo). We observed cellular UCP4 expression using fluorescence microscopy. UCP4 expression in the cell culture in response to diazoxide application was determined by a reverse transcription‐polymerase chain reaction and western blotting. Results Whereas FN stimulation increased UCP4 protein and mRNA levels, carbachol administration induced UCP4 expression in vitro and in vivo. The attenuation of this effect by atropine suggests that FN‐induced UCP4 expression involves the cholinergic pathway. The opening of mitochondrial K+ATP channels with diazoxide increased the production of ROS and led to increased UCP4 expression. In contrast, quenching ROS with superoxide dismutase reversed the effect of diazoxide on UCP4 expression. Therefore, the opening of mitochondrial K+ATP channels increased ROS production, which subsequently enhanced UCP4 expression and attenuated ROS generation. Conclusion Neuroprotective effect of FN stimulation involves activation of the cholinergic pathways, which increases ROS production by opening mitochondrial K+ATP channels, leading to increased expression of neuroprotective UCP4.

Abstract Background Skeletal muscle is mainly responsible for insulin‐stimulated glucose disposal. Dysfunction in skeletal muscle metabolism especially during obesity contributes to the insulin resistance. Astaxanthin (AX), a natural antioxidant, has been shown to ameliorate hepatic insulin resistance in obese mice. However, its effects in skeletal muscle are poorly understood. The current study aimed to investigate the molecular target of AX in ameliorating skeletal muscle insulin resistance. Methods We fed 6‐week‐old male C57BL/6J mice with normal chow (NC) or NC supplemented with AX (NC+AX) and high‐fat‐diet (HFD) or HFD supplemented with AX for 24 weeks. We determined the effect of AX on various parameters including insulin sensitivity, glucose uptake, inflammation, kinase signaling, gene expression, and mitochondrial function in muscle. We also determined energy metabolism in intact C2C12 cells treated with AX using the Seahorse XFe96 Extracellular Flux Analyzer and assessed the effect of AX on mitochondrial oxidative phosphorylation and mitochondrial biogenesis. Results AX‐treated HFD mice showed improved metabolic status with significant reduction in blood glucose, serum total triglycerides, and cholesterol (p< 0.05). AX‐treated HFD mice also showed improved glucose metabolism by enhancing glucose incorporation into peripheral target tissues, such as the skeletal muscle, rather than by suppressing gluconeogenesis in the liver as shown by hyperinsulinemic–euglycemic clamp study. AX activated AMPK in the skeletal muscle of the HFD mice and upregulated the expressions of transcriptional factors and coactivator, thereby inducing mitochondrial remodeling, including increased mitochondrial oxidative phosphorylation component and free fatty acid metabolism. We also assessed the effects of AX on mitochondrial biogenesis in the siRNA‐mediated AMPK‐depleted C2C12 cells and showed that the effect of AX was lost in the genetically AMPK‐depleted C2C12 cells. Collectively, AX treatment (i) significantly ameliorated insulin resistance and glucose intolerance through regulation of AMPK activation in the muscle, (ii) stimulated mitochondrial biogenesis in the muscle, (iii) enhanced exercise tolerance and exercise‐induced fatty acid metabolism, and (iv) exerted antiinflammatory effects via its antioxidant activity in adipose tissue. Conclusions We concluded that AX treatment stimulated mitochondrial biogenesis and significantly ameliorated insulin resistance through activation of AMPK pathway in the skeletal muscle.

Background/Aims: The migration of mesenchymal cells is a fundamental cellular process that has been implicated in many pathophysiological conditions and is induced by chemoattractants such as platelet-derived growth factors (PDGFs). However, the regulatory mechanisms shaping this migration remain to be elucidated. Methods: Here, we prepared mouse skin fibroblasts inactivated for different PDGF receptor genes and systematically measured their chemotactic responses within a gradient of different chemoattractants. Results: We found that PDGFRαβ and PDGFRββ dimers were strong inducers of random and directionally-persistent migration, respectively, that was sustained for up to 24 h. MAPK and PI3K were necessary to mediate random and directional migration, respectively. Directional migration was accompanied by abundant ventral stress fiber formation and consistent cell shape with less frequent formation of branch-like processes. Conclusion: This is the first systematic study that characterized the chemotaxis mediated by three-different types of PDGFR dimers in mesenchymal cell migration. Our data demonstrate that PDGFR dimer formation is the critical step to determine the specific mode of fibroblast chemotaxis, while the accompanying cytoskeletal remodeling might contribute to migration persistence.

Meflin (Islr) expression has gained attention as a marker for mesenchymal stem cells, but its function remains largely unexplored. Here, we report the generation of Meflin-CreERT2 mice with CreERT2 inserted under the Meflin gene promoter to label Meflin-expressing cells genetically, thereby enabling their lineages to be traced. We found that in adult mice, Meflin-expressing lineage cells were present in adipose tissue stroma and had differentiated into mature adipocytes. These cells constituted Crown-like structures in the adipose tissue of mice after high-fat diet loading. Cold stimulation led to the differentiation of Meflin-expressing lineage cells into beige adipocytes. Thus, the Meflin-CreERT2 mouse line is a useful new tool for visualizing and tracking the lineage of Meflin-expressing cells.

Pericyte desorption from retinal blood vessels and subsequent vascular abnormalities are the pathogenesis of diabetic retinopathy (DR). Although the involvement of abnormal signals including platelet-derived growth factor receptor-β (PDGFRβ) and vascular endothelial growth factor-A (VEGF-A) have been hypothesized in DR, the mechanisms that underlie this processes are largely unknown. Here, novel retinopathy mouse model (N-PRβ-KO) was developed with conditional Pdgfrb gene deletion by Nestin promoter-driven Cre recombinase (Nestin-Cre) that consistently reproduced through early non-proliferative to late proliferative DR pathologies. Depletion of Nestin-Cre-sensitive PDGFRβ+NG2+αSMA− pericytes suppressed pericyte-coverages and induced severe vascular lesion and hemorrhage. Nestin-Cre-insensitive PDGFRβ+NG2+αSMA+ pericytes detached from the vascular wall, and subsequently changed into myofibroblasts in proliferative membrane to cause retinal traction. PDGFRα+ astrogliosis was seen in degenerated retina. Expressions of placental growth factor (PlGF), VEGF-A and PDGF-BB were significantly increased in the retina of N-PRβ-KO. PDGF-BB may contribute to the pericyte-fibroblast transition and glial scar formation. Since VEGFR1 signal blockade significantly ameliorated the vascular phenotype in N-PRβ-KO mice, the augmented VEGFR1 signal by PlGF and VEGF-A was indicated to mediate vascular lesions. In addition to PDGF-BB, PlGF and VEGF-A with their intracellular signals may be the relevant therapeutic targets to protect eyes from DR. Keywords: PDGF, PlGF, VEGF, Proliferative membrane, Pathological angiogenesis, Retinopathy

We aimed to investigate whether high salt intake affects bladder function via epithelial sodium channel (ENaC) by using Dahl salt-resistant (DR) and salt-sensitive (DS) rats. Bladder weight of DR + high-salt diet (HS, 8% NaCl) and DS + HS groups were significantly higher than those of DR + normal-salt diet (NS, 0.3% NaCl) and DS + NS groups after one week treatment. We thereafter used only DR + HS and DS + HS group. Systolic and diastolic blood pressures were significantly higher in DS + HS group than in DR + HS group after the treatment period. Cystometrogram showed the intercontraction intervals (ICI) were significantly shorter in DS + HS group than in DR + HS group during infusion of saline. Subsequent infusion of amiloride significantly prolonged ICI in DS + HS group, while no intra-group difference in ICI was observed in DR + HS group. No intra- or inter-group differences in maximum intravesical pressure were observed. Protein expression levels of ENaCα in the bladder were significantly higher in DS + HS group than in DR + HS group. ENaCα protein was localized at bladder epithelium in both groups. In conclusion, high salt intake is considered to cause urinary storage dysfunction via upregulation of ENaC in the bladder epithelium with salt-sensitive hypertension, suggesting that ENaC might be a candidate for therapeutic target for urinary storage dysfunction.

Adipose tissue contains macrophages that can influence both local and systemic metabolism via the secretion of cytokines. Here, Nawaz et al. report that M2-like macrophages, present in adipose tissue, create a microenvironment that inhibits proliferation of adipocyte progenitors due to the secretion of TGF-β1

Abstract Pericytes are believed to originate from either mesenchymal or neural crest cells. It has recently been reported that pericytes play important roles in the central nervous system (CNS) by regulating blood-brain barrier homeostasis and blood flow at the capillary level. However, the origin of CNS microvascular pericytes and the mechanism of their recruitment remain unknown. Here, we show a new source of cerebrovascular pericytes during neurogenesis. In the CNS of embryonic day 10.5 mouse embryos, CD31+F4/80+ hematopoietic lineage cells were observed in the avascular region around the dorsal midline of the developing midbrain. These cells expressed additional macrophage markers such as CD206 and CD11b. Moreover, the CD31+F4/80+ cells phagocytosed apoptotic cells as functionally matured macrophages, adhered to the newly formed subventricular vascular plexus, and then divided into daughter cells. Eventually, these CD31+F4/80+ cells transdifferentiated into NG2/PDGFRβ/desmin-expressing cerebrovascular pericytes, enwrapping and associating with vascular endothelial cells. These data indicate that a subset of cerebrovascular pericytes derive from mature macrophages in the very early phase of CNS vascular development, which in turn are recruited from sites of embryonic hematopoiesis such as the yolk sac by way of blood flow.

Mural cells (pericytes and vascular smooth muscle cells) are essential for the regulation of vascular networks and maintenance of vascular integrity, but their origins are diverse in different tissues and not known in the organs that arise from the ectoderm, such as skin. Here, we show that tissue-localized myeloid progenitors contribute to pericyte development in embryonic skin vasculature. A series of in vivo fate-mapping experiments indicates that tissue myeloid progenitors differentiate into pericytes. Furthermore, depletion of tissue myeloid cells and their progenitors in PU.1 (also known as Spi1) mutants results in defective pericyte development. Fluorescence-activated cell sorting (FACS)-isolated myeloid cells and their progenitors from embryonic skin differentiate into pericytes in culture. At the molecular level, transforming growth factor-β (TGF-β) induces pericyte differentiation in culture. Furthermore, type 2 TGF-β receptor (Tgfbr2) mutants exhibit deficient pericyte development in skin vasculature. Combined, these data suggest that pericytes differentiate from tissue myeloid progenitors in the skin vasculature through TGF-β signaling.

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We aimed to evaluate mineralocorticoid receptor (MR) expression in rat bladder and the physiological role of the MR-epithelial sodium channel (ENaC) pathway in controlling bladder function in 10–12-week-old, male Sprague–Dawley rats. First, we examined the mRNA expression of MR and localization of MR and ENaC-α proteins in the urinary bladder. MR mRNA expression was observed in untreated-rat urinary bladders, and MR and ENaC-α proteins were localized in the epithelium. Next, rats were treated with vehicle (controls) or fludrocortisone (an MR agonist) for 3 days, and ENaC-α protein expression levels and bladder function were evaluated on day 4. ENaC-α protein expression was significantly higher in fludrocortisone-treated rats than in controls. In addition, cystometry was performed during intravesical infusion of saline and amiloride (an ENaC inhibitor). While intercontraction intervals (ICIs) during saline infusion were significantly shorter in the fludrocortisone group than in the controls, infusion of amiloride normalized the ICIs in the fludrocortisone group. However, no intra- or inter-group differences in maximum intravesical pressure were observed. Taken together, MR protein is localized in the rat urinary bladder epithelium, and may regulate ENaC expression and bladder afferent input. The MR-ENaC pathway may be a therapeutic target for ameliorating storage symptoms.

In the hippocampus, delayed neuronal death is normally seen in neurons of the CA1 region but not in those of the CA3 region. Astrocytes have been reported to play multiple supporting or pathological roles in neuronal functioning. While evidence indicates that astrocytes could exert neuroprotective effects following ischemia, the possible underlying mechanisms remain unclear. We aimed to investigate the roles of astrocytes in the process of delayed neuronal death following transient forebrain ischemia. L-α-aminoadipic acid (L-α-AAA), an astrocyte-selective gliotoxin, was injected into the hippocampal CA3 region of rats through a cranial window to selectively damage astrocytes. Immunofluorescence staining of glial fibrillary acidic protein (GFAP) was used to evaluate the effect of L-α-AAA on astrocyte numbers. Three days after the L-α-AAA injection, transient forebrain ischemia was induced by a modification of the four-vessel occlusion procedure. Seven days after transient forebrain ischemia, hematoxylin-eosin staining was performed to reveal the morphology of hippocampal pyramidal neurons. In rats with ischemia and reperfusion, regional cerebral blood flow (rCBF) and change in intracellular Ca2+ concentration ([Ca2+]i) were separately measured in CA1 and CA3 regions. L-α-AAA injection significantly decreased the number of astrocytes in CA3, but did not affect the pattern of rCBF changes upon ischemia/reperfusion. Seven days after transient forebrain ischemia, in rats receiving L-α-AAA, delayed neuronal death comparable with that in CA1 was observed in the CA3 region. In addition, the pattern of increase in [Ca2+]i due to transient forebrain ischemia was completely changed in the hippocampal CA3. The loss of astrocytes induced a persistent increase in [Ca2+]i in the CA3 region following transient ischemia, similar to what is observed in the CA1 region. Our study indicates that astrocytes in the hippocampal CA3 region exert neuroprotective effects following transient forebrain ischemia and act by suppressing the intracellular Ca2+ overload. Furthermore, our study will most likely provide a new therapeutic strategy for brain ischemic diseases, targeted to astrocytes.

This paper studies the condition of reducing knock intensity which is the pressure oscillation initiated by auto-ignition of the end gas. The knock intensity is thought to be decreased by suppressing the reaction rate of auto-ignition. In this study, the effect of the mixture dilution which decreases the reaction rate on the knock intensity was investigated by using a spark ignition engine. In the case of low dilution, knock was observed when the auto-ignition of the end gas occurred. When the dilution ratio was over 30%, there was the condition that the knock did not occur even if the end gas auto-ignited. The combustion with low knock intensity was observed in either case that dilution gas was inert gas or air. The knock intensity was shown as a function of the maximum temperature and the maximum pressure which affected the reaction rate although the knock intensity was affected by the composition of the mixture, load, ignition timing, auto-ignition timing of the end gas and heat release quantity of the auto-ignited mixture.

Effect of fuel-air mixture diluted by inert gas on knock intensity was investigated by using a rapid compression and expansion machine. Dilution of fuel-air mixture decreased knock intensity. As the dilution ratio became high, increase in input heating value did not affect knock intensity too much although it shortened auto-ignition delay. Knock intensity is assumed to correlate with the pressure rising rate when the auto-ignition occurs. Knock intensity was shown as a function of the maximum pressure and the maximum temperature which affect the high temperature oxidation reaction rate.

Indomethacin (IMC)-induced gastrointestinal (GI) injuries are more common in rheumatoid arthritis (RA) patients than in other IMC users, and the overexpression of nitric oxide (NO) via inducible NO synthase (iNOS) is related to the seriousness of IMC-induced GI injuries. However, sufficient strategies to prevent IMC-induced GI injuries have not yet been established. In this study, we designed dispersions of rebamipide (RBM) solid nanocrystals (particle size: 30−190 nm) by a bead mill method (RBM-NDs), and investigated whether the oral administration of RBM-NDs is useful to prevent IMC-induced GI injuries. The RBM nanocrystals were spherical and had a solubility 4.71-fold greater than dispersions of traditional RBM powder (RBM-TDs). In addition, the RBM-NDs were stable for 1 month after preparation. The RBM contents in the stomach, jejunum, and ileum of rats orally administered RBM-NDs were significantly higher than in rats administered RBM-TDs. Moreover, the oral administration of RBM-NDs decreased the NO levels via iNOS and area of the GI lesions in IMC-stimulated RA (adjuvant-induced arthritis rat) rats in comparison with the oral administration of RBM-TDs. Thus, we show that the oral administration of RBM-NDs provides a high drug supply to the GI mucosa, resulting in a therapeutic effect on IMC-induced GI injuries. Solid nanocrystalline RBM preparations may offer effective therapy for RA patients.

Abstract.: Sepsis remains the leading cause of death in critically ill patients. A major problem contributing to sepsis-related high mortality is the lack of effective medical treatment. Thus, the key goal in critical care medicine is to develop novel therapeutic strategies that will impact favorably on septic patient outcome. While it is generally accepted that sepsis is an inflammatory state resulting from the systemic response to infection, apoptosis is implicated to be an important mechanism of the death of lymphocytes, gastrointestinal and lung epithelial cells, and vascular endothelial cells associated with the development of multiple organ failure in sepsis. The pivotal role of cell apoptosis is now highlighted by multiple studies demonstrating that prevention of cell apoptosis can improve survival in clinically relevant animal models of sepsis. In this review article, we address the scientific rationale for remedying apoptotic cell death in sepsis and propose that therapeutic efforts aimed at blocking cell signaling pathways leading to apoptosis may represent an attractive target for sepsis therapy. Keywords:: sepsis, apoptosis, death receptor, acute lung injury, endothelial cell, gene therapy

Accumulating evidence indicates that cancer stem cells (CSCs) drive tumorigenesis. This suggests that CSCs should make ideal therapeutic targets. However, because CSC populations in tumors appear heterogeneous, it remains unclear how CSCs might be effectively targeted. To investigate the mechanisms by which CSC populations maintain heterogeneity during self-renewal, we established a glioma sphere (GS) forming model, to generate a population in which glioma stem cells (GSCs) become enriched. We hypothesized, based on the clonal evolution concept, that with each passage in culture, heterogeneous clonal sublines of GSs are generated that progressively show increased proliferative ability.To test this hypothesis, we determined whether, with each passage, glioma neurosphere culture generated from four different glioma cell lines become progressively proliferative (i.e., enriched in large spheres). Rather than monitoring self-renewal, we measured heterogeneity based on neurosphere clone sizes (#cells/clone). Log-log plots of distributions of clone sizes yielded a good fit (r>0.90) to a straight line (log(% total clones) = k*log(#cells/clone)) indicating that the system follows a power-law (y = xk) with a specific degree exponent (k = -1.42). Repeated passaging of the total GS population showed that the same power-law was maintained over six passages (CV = -1.01 to -1.17). Surprisingly, passage of either isolated small or large subclones generated fully heterogeneous populations that retained the original power-law-dependent heterogeneity. The anti-GSC agent Temozolomide, which is well known as a standard therapy for glioblastoma multiforme (GBM), suppressed the self-renewal of clones, but it never disrupted the power-law behavior of a GS population.Although the data above did not support the stated hypothesis, they did strongly suggest a novel mechanism that underlies CSC heterogeneity. They indicate that power-law growth governs the self-renewal of heterogeneous glioma stem cell populations. That the data always fit a power-law suggests that: (i) clone sizes follow continuous, non-random, and scale-free hierarchy; (ii) precise biologic rules that reflect self-organizing emergent behaviors govern the generation of neurospheres. That the power-law behavior and the original GS heterogeneity are maintained over multiple passages indicates that these rules are invariant. These self-organizing mechanisms very likely underlie tumor heterogeneity during tumor growth. Discovery of this power-law behavior provides a mechanism that could be targeted in the development of new, more effective, anti-cancer agents.