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Abstract Blood–brain barrier (BBB) disruption contributes to brain injury and neurological impairment. Tight junctions (TJs) and cell–cell adhesion complexes develop between endothelial cells in the brain to establish and maintain the BBB. Occludin, the first transmembrane protein identified in TJs, has received intense research interest because numerous in vitro studies have suggested its importance in maintaining BBB integrity. However, its role in maintaining BBB integrity after ischemic stroke is less clear owing to the lack of in vivo evidence. This study aimed to investigate the dynamics and function of occludin across the acute and chronic phases after stroke using occludin-deficient mice. By photochemically induced thrombosis model, the expression of occludin was decreased in brain endothelial cells from ischemic lesions. The neurological function of occludin-deficient mice was continuously impaired compared to that of wild-type mice. BBB integrity evaluated by Evans blue and 0.5-kDa fluorescein in the acute phase and by 10-kDa fluorescein isothiocyanate-labeled dextran in the chronic phase was decreased to a greater extent after stroke in occludin-deficient mice. Furthermore, occludin-deficient mice showed decreased claudin-5 and neovascularization after stroke. Our study reveals that occludin plays an important role from the acute to the chronic phase after ischemic stroke in vivo.

Abstract This paper presents the 1000 ensemble flood simulations using ensemble rainfalls simulated by 4D LETKF. The number of ensemble rainfall members is large as 1000 compared to the operational rainfall products of two-digit numbers to avoid sampling errors in the three-dimensional meteorological simulation based on chaotic theory. Using the large data set, 1000 ensemble rainfall–runoff for dam catchments and high-resolution inundation simulations of large area are carried out focusing on the Kumagawa river catchment. Herewith, the comparisons were carried out with 21-member ensemble rainfalls of an operational forecast by Japan Meteorological Agency and 100-member 4D-LETKF ensemble rainfalls simulated independent of 1000-member 4D-LETKF. At the same time, the accuracy of selective 100-member ensembles out of 1000 members is investigated. As a result, although many previous research works show a large number of ensemble simulations are necessary for three-dimensional meteorological field, the number could be reduced in the catchment-average rainfall–runoff and 2.5-dimensional inundation simulations given that the rainfall prediction has a certain level of accuracy since improving the discharge prediction accuracy with lower dimension is sometimes possible by adjusting the horizontally/vertically integrated model parameters determined by topography and soil characteristics in advance against the observed rainfall. Also, the 1000 ensembles could be classified into several patterns in horizontally accumulated 2D rainfall field. Likewise, the flood flow moves toward the low elevation area and river; thus, the resultant 2.5-dimensional flood field does not show much variety as three-dimensional meteorological simulation. The paper summarizes these studies.

We previously reported that acid-degradable methylated β-cyclodextrins (Me-β-CDs)-threaded polyrotaxanes (Me-PRXs) can induce autophagic cell death through endoplasmic reticulum (ER) stress-related autophagy, even in apoptosis-resistant cells. Hence, Me-PRXs show great potential as anticancer therapeutics. In this study, peptide-supermolecule conjugates were designed to achieve the targeted delivery of Me-PRX to malignant tumors. Arg-Gly-Asp peptides are well-known binding motifs of integrin αvβ3, which is overexpressed on angiogenic sites and many malignant tumors. The tumor-targeted cyclic Arg-Gly-Asp (cRGD) peptide was orthogonally post-modified to Me-PRX via click chemistry. Surface plasmon resonance (SPR) results indicated that cRGD-Me-PRX strongly binds to integrin αvβ3, whereas non-targeted cyclic Arg-Ala-Glu (cRGE) peptide conjugated to Me-PRX (cRGE-Me-PRX) failed to interact with integrins αvβ3. In vitro, cRGD-Me-PRX demonstrated enhanced cellular internalization and antitumor activity in 4T1 cells than that of unmodified Me-PRX and non-targeted cRGE-Me-PRX, due to its ability to recognize integrin αvβ3. Furthermore, cRGD-Me-PRX accumulated effectively in tumors, leading to antitumor effects, and exhibited excellent biocompatibility and safety in vivo. Therefore, cRGD conjugation to enhance selectivity for integrin αvβ3-positive cancer cells is a promising design strategy for Me-PRXs in antitumor therapy.

To improve the therapeutic potential of β-cyclodextrin (β-CD)-threaded acid-degradable polyrotaxanes (β-CD PRXs) in cholesterol-related metabolic disorders, we investigated the effect of carboxylation of β-CD PRXs on intracellular uptake. In this study, we established a synthetic method for the modification of carboxylalkyl carbamates on β-CD PRXs without degradation and synthesized three series of carboxyalkyl carbamate group-modified β-CD PRXs with different alkyl spacer lengths. The modification of carboxymethyl carbamate (CMC), carboxyethyl carbamate (CEC), and carboxypropyl carbamate (CPC) on the β-CD PRXs slightly reduced the interaction of the PRXs with the lipid layer model compared with the modification of 2-(2-hydroxyethoxy)ethyl carbamate (HEE-PRX), which was used in our previous studies. However, all the carboxylated β-CD PRXs showed a significantly stronger interaction with a protein model compared with HEE-PRX. The carboxylated β-CD PRXs showed significantly high intracellular uptake, through macrophage scavenger receptor A (MSR-A)-mediated endocytosis, in MSR-A-positive RAW 264.7 cells compared with HEE-PRX. Interestingly, the carboxylated β-CD PRXs also showed significantly higher intracellular uptake even in MSR-A-negative cells compared with HEE-PRX. Carboxylated β-CD PRXs are considered to strongly interact with other membrane proteins, resulting in high intracellular uptake. The length of the alkyl spacer affected the intracellular uptake levels of carboxylated PRXs, however, this relationship was varied for different cell types. Furthermore, none of the carboxylated β-CD PRXs exhibited cytotoxicity in the RAW 264.7 and NIH/3T3 cells. Altogether, carboxylation of β-CD PRXs is a promising chemical modification approach for their therapeutic application because carboxylated β-CD PRXs exhibit high cellular internalization efficiency in MSR-A-negative cells and negligible toxicity.

Abstract The V-shaped arrangement of hair bundles on cochlear hair cells is critical for auditory sensing. However, regulation of hair bundle arrangements has not been fully understood. Recently, defects in hair bundle arrangement were reported in postnatal Dishevelled-associating protein (ccdc88c, alias Daple)-deficient mice. In the present study, we found that adult Daple −/− mice exhibited hearing disturbances over a broad frequency range through auditory brainstem response testing. Consistently, distorted patterns of hair bundles were detected in almost all regions, more typically in the basal region of the cochlear duct. In adult Daple −/− mice, apical microtubules were irregularly aggregated, and the number of microtubules attached to plasma membranes was decreased. Similar phenotypes were manifested upon nocodazole treatment in a wild type cochlea culture without affecting the microtubule structure of the kinocilium. These results indicate critical role of Daple in hair bundle arrangement through the orchestration of apical microtubule distribution, and thereby in hearing, especially at high frequencies.

Abstract Cryptococcus deneoformans is an opportunistic fungal pathogen that infects the lungs via airborne transmission and frequently causes fatal meningoencephalitis. Claudins (Cldns), a family of proteins with 27 members found in mammals, form the tight junctions within epithelial cell sheets. Cldn-4 and 18 are highly expressed in airway tissues, yet the roles of these claudins in respiratory infections have not been clarified. In the present study, we analyzed the roles of Cldn-4 and lung-specific Cldn-18 (luCldn-18) in host defense against C. deneoformans infection. luCldn-18-deficient mice exhibited increased susceptibility to pulmonary infection, while Cldn-4-deficient mice had normal fungal clearance. In luCldn-18-deficient mice, production of cytokines including IFN-γ was significantly decreased compared to wild-type mice, although infiltration of inflammatory cells including CD4+ T cells into the alveolar space was significantly increased. In addition, luCldn-18 deficiency led to high K+ ion concentrations in bronchoalveolar lavage fluids and also to alveolus acidification. The fungal replication was significantly enhanced both in acidic culture conditions and in the alveolar spaces of luCldn-18-deficient mice, compared with physiological pH conditions and those of wild-type mice, respectively. These results suggest that luCldn-18 may affect the clinical course of cryptococcal infection indirectly through dysregulation of the alveolar space microenvironment.

Recent studies have shown that cellular metabolism is tightly linked to the regulation of immune cells. Here, we show that activation of cholesterol metabolism, involving cholesterol uptake, synthesis, and autophagy/lipophagy, is integral to innate immune responses in macrophages. In particular, cholesterol accumulation within endosomes and lysosomes is a hallmark of the cellular cholesterol dynamics elicited by Toll-like receptor 4 activation and is required for amplification of myeloid differentiation primary response 88 (Myd88) signaling. Mechanistically, Myd88 binds cholesterol via its CLR recognition/interaction amino acid consensus domain, which promotes the protein’s self-oligomerization. Moreover, a novel supramolecular compound, polyrotaxane (PRX), inhibited Myd88‑dependent inflammatory macrophage activation by decreasing endolysosomal cholesterol via promotion of cholesterol trafficking and efflux. PRX activated liver X receptor, which led to upregulation of ATP binding cassette transporter A1, thereby promoting cholesterol efflux. PRX also inhibited atherogenesis in Ldlr–/– mice. In humans, cholesterol levels in circulating monocytes correlated positively with the severity of atherosclerosis. These findings demonstrate that dynamic changes in cholesterol metabolism are mechanistically linked to Myd88‑dependent inflammatory programs in macrophages and support the notion that cellular cholesterol metabolism is integral to innate activation of macrophages and is a potential therapeutic and diagnostic target for inflammatory diseases.

Activation of autophagy represents a potential therapeutic strategy for the treatment of diseases that are caused by the accumulation of defective proteins and the formation of abnormal organelles. Methylated β-cyclodextrins-threaded polyrotaxane (Me-PRX), a supramolecular structured polymer, induces autophagy by interacting with the endoplasmic reticulum. We previously reported on the successful activation of mitochondria-targeted autophagy by delivering Me-RRX to mitochondria using a MITO-Porter, a mitochondria-targeted nanocarrier. The same level of autophagy induction was achieved at one-twentieth the dosage for the MITO-Porter (Me-PRX) compared to the naked Me-PRX. We report herein on the quantitative evaluation of the intracellular organelle localization of both naked Me-PRX and the MITO-Porter (Me-PRX). Mitochondria, endoplasmic reticulum and lysosomes were selected as target organelles because they would be involved in autophagy induction. In addition, organelle injury and cell viability assays were performed. The results showed that the naked Me-PRX and the MITO-Porter (Me-PRX) were localized in different intracellular organelles, and organelle injury was different, depending on the route of administration, indicating that different organelles contribute to autophagy induction. These findings indicate that the organelle to which the autophagy-inducing molecules are delivered plays an important role in the level of induction of autophagy.

Doxorubicin (DOX)-conjugated acid-degradable polyrotaxanes (PRXs) were designed as supramolecular drug carriers capable of releasing drugs in acidic cellular environments. Acid-degradable PRXs composed of α-cyclodextrin (α-CD) as a cyclic molecule, poly(ethylene glycol) (PEG) as a polymer axis, and N-triphenylmethyl (N-Trt) groups as an acid-labile stopper molecules were synthesized and DOX was conjugated with the threaded α-CDs in the PRXs. Because the acid-induced cleavage of N-Trt groups in PRXs leads to PRX dissociation, the DOX-modified α-CDs were released under acidic conditions (pH 5.0). The cytotoxicity of DOX-conjugated PRXs in colon-26 cells revealed significant cell death for DOX-conjugated PRXs after 48 h of treatment. Confocal laser scanning microscopy (CLSM) analysis revealed that the fluorescence signals derived from DOX-conjugated PRXs were observed in cellular nuclei after 48 h, suggesting that the DOX-modified α-CDs were released and accumulated in cellular nuclei. These results confirmed that acid-degradable PRXs can be utilized as drug carriers capable of releasing drug-modified α-CDs in acidic lysosomes and eliciting cytotoxicity. Overall, acid-degradable PRXs represent a promising supramolecular framework for the delivery and intracellular release of drug-modified α-CDs, and PRX–drug conjugates are expected to contribute to the development of pH-responsive drug carriers for cancer therapy.

Accumulating evidence suggests that cholesterol accumulation in leukocytes is causally associated with the development of autoimmune diseases. However, the mechanism by which fatty acid composition influences autoimmune responses remains unclear. To determine whether the fatty acid composition of diet modulates leukocyte function and the development of systemic lupus erythematosus, we examined the effect of eicosapentaenoic acid (EPA) on the pathology of lupus in drug-induced and spontaneous mouse models. We found that dietary EPA supplementation ameliorated representative lupus manifestations, including autoantibody production and immunocomplex deposition in the kidneys. A combination of lipidomic and membrane dynamics analyses revealed that EPA remodels the lipid composition and fluidity of B cell membranes, thereby preventing B cell differentiation into autoantibody-producing plasma cells. These results highlight a previously unrecognized mechanism by which fatty acid composition affects B cell differentiation into autoantibody-producing plasma cells during autoimmunity, and imply that EPA supplementation may be beneficial for therapy of lupus.

The main components of tight junctions (TJ) are claudins that polymerize and form meshwork architectures called TJ strands. Here the authors present the 3.6 Å crystal structure of murine claudin-3 and show that residue P134 causes a bending of the third transmembrane helix which affects the morphology and adhesiveness of the TJ strands.

Background & Aims: Epithelial cells are joined by tight junctions (TJs) to form a cell sheet. In the stomach, epithelial cell sheet forms an essential barrier against gastric material, including gastric acid. Although the decreased expression of stomach-type claudin-18 (stCldn18), a TJ protein, is generally observed in human gastritis and gastric cancer, its pathological roles are not fully understood. We previously reported that mice lacking stCldn18 (stCldn18-/-) exhibit gastric acid leakage through TJs, which induces active gastritis at a young age. Here, we examined the gastric pathologies in mice after long-term stCldn18 deficiency. Methods: The gastric pathologies in stCldn18-/- mice were sequentially analyzed from youth to old age, and compared to those in humans. To examine the relationship between stCldn18 deficiency-induced gastric pathologies and Wnt-dependent tumorigenesis, we generated Wnt1-overexpressing stCldn18-/- mice. Results: StCldn18-/- mice developed chronic active gastritis at middle age, with expression of the chemoattractant CCL28. At old age, 20-30% of these mice developed gastric tumors with CXCL5 expression, indicative of EMT. In this process, spasmolytic polypeptide-expressing metaplasia (SPEM) cells appeared. Increased expressions of CD44-variants, TLR2, and CXCL5 indicated age-dependent changes in cell characteristics. Some features of the stCldn18-/- mouse gastric tumorigenesis resembled H pylori-infection-related human carcinogenesis. The gastric tumorigenesis was accelerated in Wnt1-overexpressing stCldn18-/- mice, indicating that Wnt is involved in the stCldn18-/- mouse gastric tumorigenesis. Conclusions: StCldn18 deficiency induced gastric tumorigenesis in mice without H pylori infection. Our findings revealed that several signaling networks, including the cytokine-, stemness-, and Wnt-signaling pathways, may be activated under the stCldn18-deficiency-induced chronic active gastritis to accelerate the gastric tumorigenesis. Keywords: Epithelial Barrier, Tight Junction, Chronic Active Gastritis, SPEM

Background & Aims: Epithelial regeneration is essential for homeostasis and repair of the mucosal barrier. In the context of infectious and immune-mediated intestinal disease, interleukin (IL) 22 is thought to augment these processes. We sought to define the mechanisms by which IL22 promotes mucosal healing. Methods: Intestinal stem cell cultures and mice were treated with recombinant IL22. Cell proliferation, death, and differentiation were assessed in vitro and in vivo by morphometric analysis, quantitative reverse transcriptase polymerase chain reaction, and immunohistochemistry. Results: IL22 increased the size and number of proliferating cells within enteroids but decreased the total number of enteroids. Enteroid size increases required IL22-dependent up-regulation of the tight junction cation and water channel claudin-2, indicating that enteroid enlargement reflected paracellular flux–induced swelling. However, claudin-2 did not contribute to IL22-dependent enteroid loss, depletion of Lgr5+ stem cells, or increased epithelial proliferation. IL22 induced stem cell apoptosis but, conversely, enhanced proliferation within and expanded numbers of transit-amplifying cells. These changes were associated with reduced wnt and notch signaling, both in vitro and in vivo, as well as skewing of epithelial differentiation, with increases in Paneth cells and reduced numbers of enteroendocrine cells. Conclusions: IL22 promotes transit-amplifying cell proliferation but reduces Lgr5+ stem cell survival by inhibiting notch and wnt signaling. IL22 can therefore promote or inhibit mucosal repair, depending on whether effects on transit-amplifying or stem cells predominate. These data may explain why mucosal healing is difficult to achieve in some inflammatory bowel disease patients despite markedly elevated IL22 production. Keywords: Interleukin 22, Enteroid, Intestinal Stem Cell, Transit-Amplifying Cell, Regeneration, wnt, notch, tight junction, claudin-2

Hydrogels are promising materials in tissue engineering scaffolds for healing and regenerating damaged biological tissues. Previously, we developed supramolecular hydrogels using polyrotaxane (PRX), consisting of multiple cyclic molecules threaded by an axis polymer for modulating cellular responses. However, since hydrogels generally have a large amount of water, their adhesion to tissues is extremely weak. Herein, we designed a bilayered hydrogel with a PRX layer and a collagen layer (PRX/collagen hydrogel) to achieve rapid and strong adhesion to the target tissue. The PRX/collagen hydrogel was fabricated by polymerizing PRX crosslinkers in water with placement of a collagen sponge. The differences in components between the PRX and collagen layers were analyzed using Fourier transform infrared spectroscopy (FT-IR). After confirming that the fibroblasts adhered to both layers of the PRX/collagen hydrogels, the hydrogels were implanted subcutaneously in mice. The PRX hydrogel without collagen moved out of its placement site 24 h after implantation, whereas the bilayer hydrogel was perfectly adherent at the site. Together, these findings indicate that the bilayer structure generated using PRX and collagen may be a rational design for performing anisotropic adhesion.

Cyclodextrin (CD)-threaded polyrotaxanes (PRXs) with reactive functional groups at the terminals of the axle polymers are attractive candidates for the design of supramolecular materials. Herein, we describe a novel and simple synthetic method for end-reactive PRXs using bis(2-amino-3-phenylpropyl) poly(ethylene glycol) (PEG-Ph-NH2) as an axle polymer and commercially available 4-substituted benzoic acids as capping reagents. The terminal 2-amino-3-phenylpropyl groups of PEG-Ph-NH2 block the dethreading of the α-CDs after capping with 4-substituted benzoic acids. By this method, two series of azide group-terminated polyrotaxanes (benzylazide: PRX-Bn-N3, phenylazide: PRX-Ph-N3,) were synthesized for functionalization via click reactions. The PRX-Bn-N3 and PRX-Ph-N3 reacted quickly and efficiently with p-(tert-butyl)phenylacetylene via copper-catalyzed click reactions. Additionally, the terminal azide groups of the PRX-Bn-N3 could be modified with dibenzylcyclooctyne (DBCO)-conjugated fluorescent molecules via a copper-free click reaction; this fluorescently labeled PRX was utilized for intracellular fluorescence imaging. The method of synthesizing end-reactive PRXs described herein is simple and versatile for the design of diverse functional PRXs and can be applied to the fabrication of PRX-based supramolecular biomaterials.

Background: Spatial disorientation is one of the most frequent causes of aircraft accidents, and is thus a major problem affecting air safety. Although a number of studies have examined spatial disorientation, the precise physiological changes occurring as a direct result of spatial disorientation and motion sickness remain unclear. The present study sought to investigate electrodermal activity (EDA) and subjective autonomic symptoms during spatial disorientation training, and to develop an indicator of physiological changes for pilot candidates.Methods: In the current study, we investigated changes in EDA measured using a wrist-worn device, and subjective autonomic nervous system symptoms during spatial disorientation training for pilot candidates. We then used the Graybiel diagnostic criteria to develop a novel physiological biomarker.Results: We found that maximum EDA change and peak amplitude were significantly increased in participants with a Graybiel score of ≥3 points compared with those who scored < 2 points. Furthermore, for symptoms of cold sweating and saliva secretion (from the seven Graybiel diagnostic criteria), the maximum EDA change in participants with scores ≥1 point was significantly higher than that of participants scoring 0 points.Conclusion: Our results indicate that EDA data measured with a wrist-worn device could provide a useful method for objective evaluation of the severity of spatial disorientation and motion sickness.

Oral tissue regeneration has received growing attention for improving the quality of life of patients. Regeneration of oral tissues such as alveolar bone and widely defected bone has been extensively investigated, including regenerative treatment of oral tissues using therapeutic cells and growth factors. Additionally, small-molecule drugs that promote bone formation have been identified and tested as new regenerative treatment. However, treatments need to progress to realize successful regeneration of oral functions. In this review, we describe recent progress in development of regenerative treatment of oral tissues. In particular, we focus on cyclodextrin (CD)-based pharmaceutics and polyelectrolyte complexation of growth factors to enhance their solubility, stability, and bioactivity. CDs can encapsulate hydrophobic small-molecule drugs into their cavities, resulting in inclusion complexes. The inclusion complexation of osteoinductive small-molecule drugs improves solubility of the drugs in aqueous solutions and increases in vitro osteogenic differentiation efficiency. Additionally, various anionic polymers such as heparin and its mimetic polymers have been developed to improve stability and bioactivity of growth factors. These polymers protect growth factors from deactivation and degradation by complex formation through electrostatic interaction, leading to potentiation of bone formation ability. These approaches using an inclusion complex and polyelectrolyte complexes have great potential in the regeneration of oral tissues.

Niemann-Pick type C (NPC) disease is characterized by the accumulation of cholesterol in lysosomes. We have previously reported that biocleavable polyrotaxanes (PRXs) composed of β-cyclodextrins (β-CDs) threaded onto a linear polymer capped with bulky stopper molecules via intracellularly cleavable linkers show remarkable cholesterol reducing effects in NPC disease patient-derived fibroblasts owing to the stimuli-responsive intracellular dissociation of PRXs and subsequent β-CD release from the PRXs. Herein, we describe a series of novel acid-labile 2-(2-hydroxyethoxy)ethyl group-modified PRXs (HEE-PRXs) bearing terminal N-triphenylmethyl (N-Trt) groups as a cleavable component for the treatment of NPC disease. The N-Trt end groups of the HEE-PRXs underwent acidic pH-induced cleavage and led to the dissociation of their supramolecular structure. A kinetic study revealed that the number of HEE groups on the PRX did not affect the cleavage kinetics of the N-Trt end groups of the HEE-PRXs. The effect of the number of HEE groups of the HEE-PRXs, which was modified to impart water solubility to the PRXs, on cellular internalization efficiency, lysosomal localization efficiency, and cholesterol reduction ability in NPC disease-derived fibroblasts (NPC1 fibroblasts) was also investigated. The cellular uptake and lysosomal localization efficiency were almost equivalent for HEE-PRXs with different numbers of HEE groups. However, the cholesterol reducing ability of the HEE-PRXs in NPC1 fibroblasts was affected by the number of HEE groups, and HEE-PRXs with a high number of HEE groups were unable to reduce lysosomal cholesterol accumulation. This deficiency is most likely due to the cholesterol-solubilizing ability of HEE-modified β-CDs released from the HEE-PRXs. We conclude that the N-Trt group acts as a cleavable component to induce the lysosomal dissociation of HEE-PRXs, and acid-labile HEE-PRXs with an optimal number of HEE groups (4.1 to 5.4 HEE groups per single β-CD threaded onto the PRX) have great therapeutic potential for treating NPC disease.

Neuroleptic malignant syndrome (NMS) is rare but sometimes causes life-threatening conditions. We report the case of a 53-year-old male patient who developed NMS following cardiac surgery. He was diagnosed with schizophrenia and pre- scribed blonanserin, chlorpromazine, and biperiden. From postoperative day 3, hyperthermia, disturbed consciousness, and involuntary movement were observed. Subsequently, his serum creatine phosphokinase (CPK) levels increased. After NMS was suspected, chlorpromazine and biperiden were stopped. From postoperative day 7, intravenous administration of dant- rolene was initiated. Following this treatment, his serum CPK levels gradually decreased, and the other symptoms improved. The treatment of NMS remains controversial. There is no evidence that dantrolene is effective for treating NMS; however, it may be one of the important options for treating NMS. We present the case and discuss the diagnosis and management of NMS following cardiac surgery. [Arch Clin Exp Surg 2017; 6(2.000): 117-119]

Tough mechanical properties are generally required for tissue substitutes used in regeneration of damaged tissue, as these substitutes must be able to withstand the external physical force caused by stretching. Gelatin, a biopolymer derived from collagen, is a biocompatible and cell adhesive material, and is thus widely utilized as a component of biomaterials. However, the application of gelatin hydrogels as a tissue substitute is limited owing to their insufficient mechanical properties. Chemical cross-linking is a promising method to improve the mechanical properties of hydrogels. We examined the potential of the chemical cross-linking of gelatin hydrogels with carboxy-group-modified polyrotaxanes (PRXs), a supramolecular polymer comprising a poly(ethylene glycol) chain threaded into the cavity of α-cyclodextrins (α-CDs), to improve mechanical properties such as stretchability and toughness. Cross-linking gelatin hydrogels with threading α-CDs in PRXs could allow for freely mobile cross-linking points to potentially improve the mechanical properties. Indeed, the stretchability and toughness of gelatin hydrogels cross-linked with PRXs were slightly higher than those of the hydrogels with the conventional chemical cross-linkers 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS). In addition, the hysteresis loss of gelatin hydrogels cross-linked with PRXs after repeated stretching and relaxation cycles in a hydrated state was remarkably improved in comparison with that of conventional cross-linked hydrogels. It is considered that the freely mobile cross-linking points of gelatin hydrogels cross-linked with PRXs attenuates the stress concentration. Accordingly, gelatin hydrogels cross-linked with PRXs would provide excellent mechanical properties as biocompatible tissue substitutes exposed to a continuous external physical force.

Objectives: The prevalence of aortic valve disease is increasing. The purpose of this study was to compare the outcomes of patients undergoing ministernotomy versus conventional full sternotomy for aortic valve replacement (AVR). Materials and Methods: Between May 2009 and November 2013, 59 consecutive patients underwent AVR in our institution; 27 patients received upper ministernotomy (Group M), and 32 received conventional median full sternotomy (Group F). Pre-operative characteristics, aortic valve pathology, and left ventricular function in both groups were similar. Results: No differences were found in mean operation time, volume of blood loss, mean cardiopulmonary time, aortic clamp time, transfusion rate, or mortality and morbidity rates. However, the amount of red blood cells used in transfusion was significantly less in Group F than in Group M (7.3 +/- 4.5 vs. 4.6 +/- 3.3 units, P = 0.015). Conclusions: Although ministernotomy can be safely used for AVR without prolonging operative or cardiopulmonary time, no significant advantages were discerned for choosing ministernotomy over conventional full median sternotomy. [Arch Clin Exp Surg 2015; 4(2.000): 69-73]

Background Forest management strategies such as thinning have long been used to enhance ecosystem functions, especially in plantations. Thinning in plantations with high deer density, however, may not yield a desired increase in understory vegetation because deer graze on germinating plants after thinning. Here, we examine the changes in understory vegetation after thinning in plantations that have been overgrazed by sika deer to provide insight into the effects of thinning on ecosystem functions such as soil conservation and biological diversity. Methods We conducted our survey in the Tanzawa Mountains of eastern Japan. We surveyed the change in understory vegetation within and outside of three deer exclosures on a single slope with three levels of understory vegetation cover: sparse (1%, exclosure “US”), moderate (30%, exclosure “MM”), and dense (80%, exclosure “LD”) over 10 years after a 30% thinning of an old-growth cedar and cypress plantation which was overgrazed by sika deer. Results Understory vegetation cover, biomass and species richness increased within and outside the “US” and “MM” exclosures after thinning, and biomass was greater within than outside the exclosures at 10 years after thinning. Unpalatable species dominated both “US” and “MM” exclosures before thinning, and trees and shrubs dominated within the exclosures over time after thinning. In contrast, unpalatable, grazing-tolerant, perennial, and annual species increased outside the “US” and “MM” exclosures. No noticeable changes were observed within and outside the “LD” exclosure when compared with the “US” and “MM” exclosures. Conclusions Our results suggest that thinning a stand by 30% based on volume resulted in an increase in understory vegetation cover mainly composed of both unpalatable and grazing-tolerant species in a plantation forest where understory vegetation is sparse or moderate and sika deer density is high. We emphasize that establishing deer exclosures or controlling deer is essential to maintaining similar understory vegetation both within and outside exclosures.

Bone morphogenetic protein 2 (BMP-2) has received considerable attention because of its osteoinductivity, but its use is limited owing to its instability and adverse effects. To reduce the dose of BMP-2, complexation with heparin is a promising approach, because heparin enhances the osteoinductivity of BMP-2. However, the clinical use of heparin is restricted because of its anticoagulant activity. Herein, to explore alternative polymers that show heparin-like activity, four polycarboxylates, poly(acrylic acid) (PAA), poly(methacrylic acid) (PMAA), poly(aspartic acid) (PAsp), and poly(glutamic acid) (PGlu), were selected and their capability to modulate the osteoinductivity of BMP-2 was evaluated. Dynamic light scattering indicated that these polycarboxylates formed polyelectrolyte complexes with BMP-2. The osteogenic differentiation efficiency of MC3T3-E1 cells treated with the polycarboxylate/BMP-2 complexes was investigated in comparison to that of the heparin/BMP-2 complex. As a result, PGlu/BMP-2 complex showed the highest activity of alkaline phosphatase, which is an early-stage marker of osteogenic differentiation, and rapid mineralization. Based on these observations, PGlu could serve as an alternative to heparin in the regenerative therapy of bone using BMP-2.

Macrophages play an important role in the regulation of inflammation and immune response as well as the pathogenesis of chronic inflammatory diseases and cancer. Therefore, targeted delivery of therapeutic reagents to macrophages is an effective method for treatment and diagnosis. We previously examined the therapeutic applications of polyrotaxanes (PRXs) comprised of multiple cyclodextrins (CDs) threaded on a polymer chain and capped with bulky stopper molecules. In the present study, we designed an α-d-mannose-modified α-CD/poly(ethylene glycol)-based PRX (Man-PRX). The intracellular uptake of Man-PRX through the interaction with macrophage mannose receptor (MMR) in macrophage-like RAW264.7 cells was examined. Intracellular Man-PRX uptake was observed in MMR-positive RAW264.7 cells but was negligible in MMR-negative NIH/3T3 cells. In addition, the intracellular Man-PRX uptake in RAW264.7 cells was significantly inhibited in the presence of free α-d-mannose and an anti-MMR antibody, which suggests that MMR is involved in the intracellular uptake of Man-PRX. Moreover, the polarization of RAW264.7 cells affected the Man-PRX internalization efficiency. These results indicate that Man-PRX is an effective candidate for selective targeting of macrophages through a specific interaction with the MMR.

Helicobacter cinaedi is a rare human pathogen which has various clinical manifestations such as cellulitis, bacteremia, arthritis, meningitis, and infectious endocarditis. We report an abdominal aortic aneurysm infected with Helicobacter cinaedi, treated successfully with surgical repair and long-term antimicrobial therapy.

Abstract.: After the report of the Cardiac Arrhythmia Suppression Trial, a tabular framework of the Sicilian Gambit has been proposed to display actions of antiarrhythmic drugs on ion channels and receptors and to provide more rational pharmacotherapy of arrhythmias. However, because effects of antiarrhythmic drugs on If have not been thoroughly examined, we used patch clamp techniques to determine the effects of various antiarrhythmic drugs on the HCN (hyperpolarization-activated cyclic nucleotide–gated) channel currents. HCN4 channels, a dominant isoform of HCN channels in the heart, were expressed in HEK293 cells. Amiodarone and bepridil potently inhibited the HCN4 channel current with IC50 values of 4.5 and 4.9 μM, respectively, which were close to their therapeutic concentrations. The inhibitory effects of quinidine, disopyramide, cibenzoline, lidocaine, mexiletine, aprindine, propafenone, flecainide, propranolol, and verapamil on the HCN4 channel current were weak in their therapeutic concentrations, with IC50 values of 78.3, 249, 46.8, 276, 309, 43.7, 14.3, 1700, 50.5, and 44.9 μM, respectively, suggesting that the inhibitory effects on If would be clinically small. d,l-Sotalol hardly affected the HCN4 channel current. Information about the HCN4-channel effects of many antiarrhythmic drugs may be useful for determining the appropriate drug for treatment of various arrhythmias while minimizing adverse effects. Keywords:: antiarrhythmic drug, hyperpolarization-activated cyclic nucleotide–gated (HCN) channel subtype 4, pacemaker current, amiodarone, bepridil

The flow field of an offset jet is complex and is frequently found in many engineering devices. In this paper, the fluid flow and heat transfer characteristics of two-dimensional offset jet are investigated experimentally. The offset jet is produced by the flow of air which issue from the end of a long parallel channel. The exit Reynolds number Re is changed in 1.5×103 to 7.0×103, and the offset ratio H/h (H : step height, h : channel height) is changed in 0.5, 1.0 and 1.5. The wall shear stress is measured using a micro flow sensor. Flow visualization is carried out using a CCD camera and laser light. The velocity profiles are measured by a PIV system. When the flow at the channel exit is laminar, fluid in the outer region has a counter-clockwise vorticity and fluid in the inner region has a clockwise vorticity, and a lifting-off of the wall jet flow into the ambient fluid is occurred. The Strouhal number of the vortex frequency is almost constant against the offset ratio. The formation of those vortices is not seen at the turbulent flow. Two peaks exist in the wall static pressure, Nusselt number and skin friction coefficient distributions of the laminar flow with H/h = 0.5 and 1.0. The 1st peak appears near the reattachment point, and the 2nd peak appears in the neighborhood of the lifting-off of the wall jet flow. The two peaks coalesce into one at H/h = 1.5. As the offset ratio increases, the reattachment point moves to downstream direction and Nu becomes large both the laminar and turbulent flows. The reattachment point of the turbulent flow moves to upstream direction compared with the laminar flow because Coanda-effect is strengthen.

The striatum plays an important role in linking cortical activity to basal ganglia outputs. Group I metabotropic glutamate receptors (mGluRs) are densely expressed in the medium spiny projection neurons and may be a therapeutic target for Parkinson's disease. The group I mGluRs are known to modulate the intracellular Ca(2+) signaling. To characterize Ca(2+) signaling in striatal cells, spontaneous cytoplasmic Ca(2+) transients were examined in acute slice preparations from transgenic mice expressing green fluorescent protein (GFP) in the astrocytes. In both the GFP-negative cells (putative-neurons) and astrocytes of the striatum, spontaneous slow and long-lasting intracellular Ca(2+) transients (referred to as slow Ca(2+) oscillations), which lasted up to approximately 200 s, were found. Neither the inhibition of action potentials nor ionotropic glutamate receptors blocked the slow Ca(2+) oscillation. Depletion of the intracellular Ca(2+) store and the blockade of inositol 1,4,5-trisphosphate receptors greatly reduced the transient rate of the slow Ca(2+) oscillation, and the application of an antagonist against mGluR5 also blocked the slow Ca(2+) oscillation in both putative-neurons and astrocytes. Thus, the mGluR5-inositol 1,4,5-trisphosphate signal cascade is the primary contributor to the slow Ca(2+) oscillation in both putative-neurons and astrocytes. The slow Ca(2+) oscillation features multicellular synchrony, and both putative-neurons and astrocytes participate in the synchronous activity. Therefore, the mGluR5-dependent slow Ca(2+) oscillation may involve in the neuron-glia interaction in the striatum.

Neuronal morphogenesis is implicated in neuronal function and development with rearrangement of cytoskeletal organization. Ezrin, a member of Ezrin/Radixin/Moesin (ERM) proteins links between membrane proteins and actin cytoskeleton, and contributes to maintenance of cellular function and morphology. In cultured hippocampal neurons, suppression of both radixin and moesin showed deficits in growth cone morphology and neurite extensions. Down-regulation of ezrin using siRNA caused impairment of netrin-1-induced axon outgrowth in cultured cortical neurons. However, roles of ezrin in the neuronal morphogenesis of the cultured neurons have been poorly understood. In this report, we performed detailed studies on the roles of ezrin in the cultured cortical neurons prepared from the ezrin knockdown (Vil2(kd/kd)) mice embryo that showed a very small amount of ezrin expression compared with the wild-type (Vil2(+/+)) neurons. Ezrin was mainly expressed in cell body in the cultured cortical neurons. We demonstrated that the cultured cortical neurons prepared from the Vil2(kd/kd) mice embryo exhibited impairment of neuritogenesis. Moreover, we observed increased RhoA activity and phosphorylation of myosin light chain 2 (MLC2), as a downstream effector of RhoA in the Vil2(kd/kd) neurons. In addition, inhibition of Rho kinase and myosin II rescued the impairment of neuritogenesis in the Vil2(kd/kd) neurons. These data altogether suggest a novel role of ezrin in the neuritogenesis of the cultured cortical neurons through down-regulation of RhoA activity.

Ezrin is a membrane-associated cytoplasmic protein that serves to link cell-membrane proteins with the actin-based cytoskeleton, and also plays a role in regulation of the functional activities of some transmembrane proteins. It is expressed in placental trophoblasts. We hypothesized that placental ezrin is involved in the supply of nutrients from mother to fetus, thereby influencing fetal growth. The aim of this study was firstly to clarify the effect of ezrin on fetal growth and secondly to determine whether knockout of ezrin is associated with decreased concentrations of serum and placental nutrients. Ezrin knockout mice (Ez(-/-)) were confirmed to exhibit fetal growth retardation. Metabolome analysis of fetal serum and placental extract of ezrin knockout mice by means of capillary electrophoresis-time-of-flight mass spectrometry revealed a markedly decreased concentration of hypotaurine, a precursor of taurine. However, placental levels of cysteine and cysteine sulfinic acid (precursors of hypotaurine) and taurine were not affected. Lack of hypotaurine in Ez(-/-) mice was confirmed by liquid chromatography with tandem mass spectrometry. Administration of hypotaurine to heterogenous dams significantly decreased the placenta-to-maternal plasma ratio of hypotaurine in wild-type fetuses but only slightly decreased it in ezrin knockout fetuses, indicating that the uptake of hypotaurine from mother to placenta is saturable and that disruption of ezrin impairs the uptake of hypotaurine by placental trophoblasts. These results indicate that ezrin is required for uptake of hypotaurine from maternal serum by placental trophoblasts, and plays an important role in fetal growth.

The sodium hydrogen exchanger isoform 3 (NHE3) mediates absorption of sodium, bicarbonate and water from renal and intestinal lumina. This activity is fundamental to the maintenance of a physiological plasma pH and blood pressure. To perform this function NHE3 must be present in the apical membrane of renal tubular and intestinal epithelia. The molecular determinants of this localization have not been conclusively determined, although linkage to the apical actin cytoskeleton through ezrin has been proposed. We set out to evaluate this hypothesis. Functional studies of NHE3 activity were performed on ezrin knockdown mice (Vil2(kd/kd)) and NHE3 activity similar to wild-type animals detected. Interpretation of this finding was difficult as other ERM (ezrin/radixin/moesin) proteins were present. We therefore generated an epithelial cell culture model where ezrin was the only detectable ERM. After knockdown of ezrin expression with siRNA, radixin and moesin expression remained undetectable. Consistent with the animal ultrastructural data, cells lacking ezrin retained an epithelial phenotype but had shortened and thicker microvilli. NHE3 localization was identical to cells transfected with non-targeting siRNA. The attachment of NHE3 to the apical cytoskeleton was unaltered as assessed by fluorescent recovery after photobleaching (FRAP) and the solubility of NHE3 in Triton X-100. Baseline NHE3 activity was unaltered, however, cAMP-dependent inhibition of NHE3 was largely lost even though NHE3 was phosphorylated at serines 552 and 605. Thus, ezrin is not necessary for the apical localization, attachment to the cytoskeleton, baseline activity or cAMP induced phosphrylation of NHE3, but instead is required for cAMP mediated inhibition.

The immunostimulatory effects of the representative dental resin monomer 2-hydroxyethyl methacrylate (HEMA), a HEMA derivative that does not contain a double bond (2-hydroxyethyl isobutyrate, HEIB), and polymerized water-soluble oligomers of HEMA (PHEMA) were investigated. It is known that expression levels of either or both of CD54 and CD86 in THP-1 cells are increased by exposure to sensitizing substances. In this study, the expression levels of CD54 and CD86, the production of reactive oxygen species (ROS), and the viability of the cells were measured after 24 h of incubation with these materials at different concentrations. The concentrations of the materials that induced the expression of both CD54 and CD86 were low in the following order: NiSO4, HEMA, and methyl methacrylate (MMA). These results indicate that these dental resin monomers have lower sensitizing potentials than NiSO4. Although HEIB, which lacks a double bond, resulted in negligible ROS production and reduced cytotoxicity than HEMA, it induced the expression of CD54 and CD86. Comparison of the results for HEMA and HEIB indicates that dental resin monomer-induced sensitization may be related not only to the oxidative stress related to the methacryloyl group but also to the structures of these compounds. Of particular interest is the result that a water-soluble PHEMA oligomer with a relatively high-molecular weight also exhibited negligible cytotoxicity, whereas the expression level of CD54 increased after exposure to PHEMA at a high concentration. This result serves as a warning that polymerized substances also have the potential to induce sensitization. This study provides insight into the nature of allergic responses to dental resin materials in clinical use and may facilitate the development of more biocompatible restorative materials in the future.