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Trifecta valve is a 3rd-generation, stented bioprosthesis which is made from one bovine pericardial sheet. A 77-years-old male patient had undergone combine aortic valve replacement (AVR) using a 23-mm Trifecta valve and ascending aorta replacement for severe aortic valve regurgitation and ascending aorta aneurysm. The postoperative period was uneventful. However, he presented with dyspnea on effort and severe aortic valve regurgitation 31 months after operation. Re-do AVR with a new bioprosthetic valve was performed via 2nd sternotomy. Surgical inspection revealed that the Trifecta valve had a parastent tear in the left-coronary cusp without any calcification or vegetation. We report our experience with a case of early structural valve deterioration of Trifecta biological prosthesis and review relevant literatures.

Objective: We used the subjective visual vertical (SVV) and two different subjective visual body axis (SVBA) methods to quantify roll-tilt perception under gravity, and investigated the characteristics of these methods during static roll-tilt. In addition, we independently developed a compact device to facilitate evaluation of SVBA in different gravitational environments.Methods: Ten male volunteers participated in this study. We created a roll-tilt environment using a flight simulator in a dark room. The cockpit of the simulator was tilted leftward or rightward (−30°, −20°, −10°, 0°, 10°, 20° and 30°) in each randomly ordered trial. We quantified roll-tilt perception such that the experiment was conducted under 21 different conditions per participant.Results: We found no significant differences among the SVV error and the two types of SVBA error.Conclusions: The SVV and the SVBA methods may be useful for evaluating subjective roll-tilt perception.

The claudins are a family of membrane proteins with at least 27 members in humans and mice. The extracellular regions of claudin proteins play essential roles in cell-cell adhesion and the paracellular barrier functions of tight junctions (TJs) in epithelial cell sheets. Furthermore, the extracellular regions of some claudins function as paracellular channels in the paracellular barrier that allow the selective passage of water, ions, and/or small organic solutes across the TJ in the extracellular space. Structural analyses have revealed a common framework of transmembrane, cytoplasmic, and extracellular regions among the claudin-based paracellular barriers and paracellular channels; however, differences in the claudins' extracellular regions, such as their charges and conformations, determine their properties. Among the biological systems that involve fluid flow and metabolism, it is noted that hepatic bile flow, renal Na+ reabsorption, and intestinal nutrient absorption are dynamically regulated via site-specific distributions of paracellular channel-forming claudins in tissue. Here, we focus on how site-specific distributions of claudin-2- and claudin-15-based paracellular channels drive their organ-specific functions in the liver, kidney, and intestine.

Epithelial cell sheet formation is central to many aspects of vertebrate development and function. For example, it is a major principle of differentiation in embryogenesis and regeneration, enables the compartmentalization of tissues, and is the basis for the maintenance of homeostasis throughout the body. A key characteristic of biologically functional epithelial cell sheets is a clear difference between the top and bottom sides owing to the apicobasal polarity of the cells in the sheet, as seen in the simple polar epithelia. Epithelial cell sheets are formed by cell-cell adhesion conferred by junctional complexes, in particular via tight junctions (TJs), which thus create a paracellular barrier. This review focuses on the apical side of the sheet, which serves as the front line. The apical membranes and TJs of the various tissues have specific characteristics that enable them to function and adapt to their biological context: each system must be robust, but also dynamic and flexible to maintain homeostasis. Here, we describe various apical cytoskeletal structures that are critical to the integrity of epithelial cell sheets. We also discuss the association of apical cytoskeletal networks with TJs, which thus forms a combined system, tentatively termed the TJ-apical complex.

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.

We report the effects of 10 years of fencing under a gap and under a closed canopy on the regeneration of tree seedlings in an old-growth fir forest overbrowsed by sika deer in the Tanzawa Mountains. We examined tree seedling emergence and growth of 10 major species over 10 years at four plots: inside and outside a deer exclosure under a gap and under a closed canopy (the plots were named “GAPF”, “GAP”, “CCF” and “CC”, respectively). Seedlings of Abies firma, Acer palmatum and Zelkova serrata were noted at all four plots during the initial investigation. Seedlings of five pioneer species emerged in GAPF and GAP, but had showed almost no emergence in CC and CCF. Seedlings of 10 target species showed the greatest tendency toward survival at GAPF among the plots. The survival of six species’ seedlings was significantly higher (p 150 cm, while A. firma, A. palmatum and Z. serrata were mainly

We developed stimuli-labile polyrotaxanes (PRXs) composed of β-cyclodextrin (β-CD), Pluronic as an axle polymer, and acid-cleavable N-triphenylmethyl groups as bulky stopper molecules, and found that the PRXs are potent therapeutics for Niemann-Pick type C disease, because the PRX can effectively reduce intracellular cholesterol through the intracellular release of threaded β-CDs. In general, the PRXs need to be chemically modified with hydrophilic functional groups because PRXs are not soluble in aqueous media. Herein, four series of oligo(ethylene glycol)s (OEGs) with different ethylene glycol repeating unit (2 or 3) and chemical structure of OEG terminal (hydroxy or methoxy) were modified onto the threaded β-CDs in PRX. The effects of the structure of OEG on the aqueous solubility, toxicity, and cellular internalization efficiency of OEG-modified PRXs were investigated to optimize the chemical structure of OEG. The hydroxy-terminated OEG-modified PRXs showed excellent solubility in aqueous media and no toxicity, regardless of the number of ethylene glycol repeating units. In the case of the methoxy-terminated OEG-modified PRXs, sufficient solubility in aqueous media and negligible toxicity were observed when the number of ethylene glycol repeating units was 3, while low solubility and toxicity were observed when the ethylene glycol repeating unit was 2. Additionally, cellular uptake levels of methoxy-terminated OEG-modified PRXs in RAW264.7 cells were higher than those of hydroxy-terminated OEG-modified PRXs. Consequently, the chemical structure of the OEG strongly affects the chemical and biological properties of the PRXs, and that a methoxy-terminated OEG with 3 ethylene glycol repeating units is the most preferable modification of PRXs, since the resultant PRX is sufficiently soluble in aqueous media, non-toxic, and possesses high cellular internalization efficiency.

In order to prevent postoperative adhesion and the related complications, a thermally crosslinked gelatin (TCG) film was developed and the basic biological properties were examined, paying special attention to the relationship between these properties and the extent of crosslinking of the film. The gelatin films crosslinked thermally for five different time periods (0, 1, 3, 8, and 14 hours) were developed and the following tests were performed. Regarding the material characterization of the films, the water content, the water solubility, and the enzymatic degradation for collagenase were found to be closely related to the duration of thermal crosslinking. In an in vitro study conducted to examine the cell growth of fibroblasts cultured on the films, the degree of cell growth, except no crosslinked film, was less than that observed in the control group, thus suggesting that such effects of the films on fibroblast cell growth may be related with their anti-adhesive effects. In in vivo tests, the films crosslinked for longer time periods (3, 8, and 14 hours) were retained for longer after being implanted into the abdominal cavity in rats and showed a significant anti-adhesive effect in the rat cecum adhesion models, indicating that the biodegradability and anti-adhesive effects of the TCG films depend on the duration of thermal crosslinking. In order to develop useful and effective anti-adhesive gelatin film, it is very important to optimize duration of the thermal crosslinking.

The ethylene glycol-based dimethacrylates are commonly used in biomaterials and dental restorative materials as a cross-linking agent. In this study, toxic effect of triethylene glycol dimethacrylate (TEGDMA) and poly(ethylene glycol) dimethacrylates (PEG-DMAs) with various ethylene glycol repeating units was investigated in terms of cytotoxicity, oxidative stress, and the expression of co-stimulatory factors in human leukemia cell line (THP-1 cells) to verify the effect of ethylene glycol repeating units. Note that the 1-octanol/water partition coefficient of PEG-based dimethacrylates decreased with increasing the ethylene glycol repeating units, indicating that the hydrophilicity of PEG-DMAs increased with ethylene glycol repeating units. The toxic effect of PEG-DMAs such as cytotoxicity, oxidative stress, and the expression of CD86 in treated THP-1 cells are reduced with increasing the ethylene glycol repeating units in PEG-DMAs. However, the expression of CD54 in treated THP-1 cells was not influenced with the ethylene glycol repeating units and the maximal expression level of CD54 was observed at the concentration range of 2-4 mM for all samples. Accordingly, hydrophilic character of PEG-DMAs with long ethylene glycol chains definitely alleviates the some toxic aspect of PEG-based DMAs. This finding would provide important insight into the design of new biomaterials and dental materials with superior biocompatibility.

The development of drugs and medical devices is necessary for medical progress; however, safety measures need to be put in place to protect the health of the population. In order to ensure the safety of drugs and medical devices, it is important to determine measures for appropriate management of risks at any time during the development phase, the regulatory review and the post-marketing phase. Adverse events detected in clinical trials are limited due to the restricted numbers of patients enrolled in the trials. Therefore, it is almost impossible to predict rare serious adverse events during the post-marketing phase. The revised Pharmaceutical Affairs Act was established in Japan in November 20, 2013. The new act focuses on increased safety of drugs and medical devices. The Pharmaceuticals and Medical Devices Agency (PMDA) is the regulatory authority in Japan that promotes safety measures from the development phase through to the post-marketing phase. In the post-marketing phase, the PMDA collects information from the medical product companies and healthcare professionals, as well as instructing and advising them with regard to post-marketing safety measures for each drug and medical device. Since Japan has a national health insurance system, a new drug or a medical device is available throughout the country when the drug price or medical fee is listed in the National Health Insurance price list. Healthcare professionals in medical institutions must learn about the drugs and medical devices they handle, and should make an effort to maintain patient safety. The PMDA medi-navi is a very useful electronic mail delivery service that provides critical information for protecting patients from health hazards caused by adverse events. The 'risk management plan' is also important as it contains important information about safety profile and post-marketing measures of a new drug.

Polyrotaxane (PRX)-containing triblock copolymers are a unique class of supramolecular polymers. In combination with the intrinsic properties of polymer chains, the supramolecular properties of PRXs, such as the freely mobile character of threading α-cyclodextrins (α-CDs) and the intermolecular hydrogen bonding, can be utilized as biomaterials. However, it is difficult to synthesize well-defined PRX-containing triblock copolymers with regulated polymeric chain length and the number of threading α-CDs. Herein, we described the precise synthetic method of PRX-containing triblock copolymers via reversible addition–fragmentation chain transfer (RAFT) polymerization using a novel PRX macro-chain transfer agent (CTA) with terminal hooks of phenylalanyl groups. The terminal phenylalanyl groups of PRX macro-CTA act as a hook to inhibit the dethreading of α-CDs during polymerization to achieve the regulation of molecular weight of the polymer chains while maintaining the number of threading α-CDs in the PRX segments. Additionally, we first prepared self-assembled polymeric micelles with an outermost PRX layer using the PRX-containing triblock copolymers. The hydroxyethyl group-modified triblock copolymers composed of PRX and hydrophobic poly(benzyl methacrylate) (PBzMA) were found to form polymeric micelles 47 nm in diameter and with a narrow size distribution. The supramolecular polymeric micelles show a core–shell-type structure comprising a core of hydrophobic PBzMA surrounded by the PRX flower loops. This micelle formation allows the incorporation of water-insoluble anticancer drugs within the PBzMA core as well as biological ligands into the α-CDs of the PRX flowers toward receptor proteins of target cell membranes. Finally, it is expected that the obtained polymeric micelles with the outermost PRX layer could be applied as a supramolecular drug carrier.

This paper aims to overview BIM (building information modeling) implementation in Singapore and to clarify the problems concerned with BIM in Singapore and Japan. The Singapore BIM Roadmap and related policies developed by BCA (Building and Construction Authority) are introduced at first along with the background and environment in Singapore. Despite of the assessment from Japan, BIM implementation in Singapore is not always perfect and well-equipped according to the authors’ interviews to Japanese general contractors operating in Singapore. Japanese general contractors have ability to produce and coordinate design drawings and shop drawings by hiring in-house architects and engineers. The client and the architects can reduce project risk concerning design and drawings by transferring it to the general contractor. The authors discuss the problems on project management to show the strategy to develop the new version of BIM, hoping to share the value with all of the stakeholders of the project.

A 17-year-old high school boy who complained of severe positional vertigo visited our hospital. He belonged to an American football team in high school, and his head often hit opponents during games. The vertigo was strongest when his left ear was down in the supine position. Physical examination and computed tomography revealed no abnormalities. However, positional testing revealed apogeotropic direction-changing horizontal nystagmus. Rightward nystagmus in the left-ear-down supine position was stronger than leftward nystagmus in the right-ear-down supine position. Additionally, in the supine position, a null point for horizontal nystagmus was identified, beyond which the nystagmus changed direction. This null point was evident when the head was turned 10 degrees to the side. The diagnosis was right lateral canal type of BPPV caused by cupulolithiasis. He was treated with the Gufoni maneuver and reported a decreased incidence of positional vertigo upon re-evaluation.

Cingulin phosphorylation by AMPK promotes its binding to α-tubulin and is required for the association of planar apical microtubules with epithelial tight junctions., Epithelial cells characteristically have noncentrosomal microtubules that are arranged in the apicobasal direction. In this paper, we examined cell sheets formed by an epithelial (Eph4) cell line by structure illumination microscopy and found a previously not clearly described planar apical network of noncentrosomal microtubules (MTs) in which the sides of the MT bundles were associated with tight junctions (TJs). In a gel overlay assay with taxol-stabilized MTs, cingulin showed strong binding to MTs, and a domain analysis showed that this binding occurred through cingulin’s N-terminal region. The association of planar apical MTs with TJs was compromised by cingulin knockdown (KD) or the expression of dephosphomimetic mutants of cingulin at its adenosine monophosphate–activated protein kinase (AMPK) target sites, whereas phosphorylation at these sites facilitated cingulin–tubulin binding. In addition, although wild-type colonies formed spheres in 3D culture, the cingulin KD cells had anisotropic shapes. These findings collectively suggest that the regulated cingulin–MT association has a specific role in TJ-related epithelial morphogenesis that is sensitive to metabolic homeostasis-related AMPK activity.

The effects of the supramolecular polyrotaxane (PRX) structure on cellular internalization are investigated by flow cytometry and confocal laser scanning microscopy. AF-545-labeled aminated PRXs (APRXs) containing different numbers of threaded α-cyclodextrins (CDs) and amino groups are synthesized; their cellular uptakes are analyzed using HeLa cells in serum. The APRX threaded CD number is discovered to be a more critical factor for enhancing cellular internalization than the APRX amine content. Additionally, APRXs are demonstrated to be more easily internalized than conventional linear cationic macromolecules. Because increased numbers of threaded CDs are related to increased PRX rigidity, the PRX rigid frame resulting from CD molecules threaded on a poly(ethylene glycol) (PEG) chain is suitable for intracellular tools in therapy and diagnosis.

ADSCs(脂肪組織由来幹細胞)は再生医療において有用な細胞源であり、臨床でも応用されている。しかしながら三次元的マトリックスの足場なしでは複雑な構造の組織再生は難しく、ADSCsを用いてこのような組織再生を行なう際には足場が必要である。従来、人工足場としてはPGA不織布が汎用されているが、PGAは疎水性であるためにADSCsの接着性と増殖性が低いと考えられる。細胞接着性と増殖性を改善するために、我々はコラーゲンコーティングPGA不織布を開発した。ラットの鼠蹊部より採取した脂肪組織から得た細胞の内でフローサイトメトリーによりCD29、90が陽性かつCD 11b、45が陰性と特徴つけられる細胞群をADSCsとして実験を行った。In Vitroの細胞増殖試験で、コラーゲンコーティングPGA不織布上では、それに対応するコラーゲン非コーティングPGA不織布上よりも細胞の接着・増殖が良好であった。この結果は、コラーゲンコーティングによりPGA不織布繊維表面が親水性になり、ADSCsの接着・増殖に有利な環境に変化したためと考えられる。, Adipose-derived stem cells (ADSCs) have attracted attention as a useful cell source for tissue engineering, even in the clinical setting. However, it is difficult to regenerate tissues with complex structures in the absence of a three-dimensional matrix such as an artificial scaffold. Namely, scaffolds are required to allow implanted ADSCs to regenerate tissues with three-dimensional structures. Although non-woven PGA (poly (glycolic acid)) fabric has been used widely as an artificial scaffold, ADSCs poorly adhere to and grow on PGA fabric due to its hydrophobicity. To improve cell adhesion and growth on PGA fibers, we devised a PGA fabric coated with collagen. We isolated ADSCs from the inguinal adipose tissue of a rat and characterized the cells using flow cytometry with both CD29- and CD90-positive markers and CD11b- and CD45-negative markers. Our cell growth examination revealed that ADSC growth was improved on all of the collagen-coated fabrics compared with that observed on the corresponding non-coated fabrics. This may be because ADSCs are able to adhere and grow more readily on the hydrophilic surface of collagen-coated PGA fabrics.

膵液漏局所化のためのポリグリコール酸不織布(PGA不織布)を開発した。膵液に対する防御壁としての肉芽組織をスムーズに密に形成する足場として、3種類の足場材料を検討した。すなわちpoly (L-lactide-ε-caprolactone)コポリマーフィルム、繊維径0.9μmのPGA不織布、繊維径20μmのPGA不織布である。ラットの膵臓表面を電気メスで焼灼する膵液漏出モデルを用いた。3種類の足場材料の各1種類の材料を膵臓の焼灼された表面を覆うように周囲組織に逢着した。術後5日間、ラットの生死を観察し、5日目に剖検して肉眼的、顕微鏡的に観察した。PGA不織布の2群では、その他の2群に比べて生存率は高く、フィルム群との間で有意差を認めた。肉眼的観察では、腹水貯留、汎発性腹膜炎や腹腔内全臓器が一塊となる腫瘤形成のような重度の病理所見がみられたが、フィルム群に対してPGA不織布の2群は有意に少なかった。顕微鏡的観察では、肉芽組織は繊維径0.9と20μmのPGA不織布の全層にわたり形成され、組織障害や炎症反応は不織布外側では内側に比べ軽度であった。このようにPGA不織布では膵液漏の局所化が可能であった。対して補強なし群とフィルム群では、組織障害や炎症反応は膵臓から離れた広範囲に及んでいた。フィルム群では特に、フィルム外側でも炎症性単核細胞の集積がフィルムに沿い広がり、フィルム周囲では防御壁としての肉芽形成は乏しかった。このように補強なし群と比較しても、フィルム群では、膵液による組織損傷や炎症がより広範囲に及び、防御壁となる肉芽形成は悪かった。結論として、PGA不織布は膵液漏の治療の足場材料として有効と考えられた。, We developed sheets of non-woven poly (glycolic acid) PGA fabric that can act as a scaffold to form smoothly dense granulation tissue for barrier walls to localize leakage of pancreatic juice. In this study, we tested three kinds of materials: poly (L-lactide-ε-caprolactone) (copolymer) film, PGA fabric-0.9 with a mean fiber diameter of 0.9µm and PGA fabric-20 with a mean fiber diameter of 20 µm. Under general anesthesia, the pancreatic surface of in rats was cauterized with an electric scalpel. Then, the pancreas was wrapped with one of the three kinds of sheets and the sheet was fixed onto the surrounding tissues. Survival was observed for five days after surgery. The survival rates were higher in the two groups of fabric-treated rats than in the other groups, although the differences were significant only for the film-treated group. Macroscopically, severe pathologic findings such as accumulation of ascites, tumor formation in whole intraperitoneal organs and generalized peritonitis occurred at a significantly lower incidence in the two fabric-treated groups than in the film-treated group. Microscopically, granulation tissue was formed along the fabrics throughout the full thickness of the fabric scaffolds of fabric-0.9 and fabric-20. In these groups, injurious tissue changes and inflammatory reactions were reduced in the outer zone of the fabric wall compared to that observed in the inner zone. Therefore, the new PGA fabrics successfully localized pancreatic juice leakage. In contrast, in the no treatment group and the film-treated group, injurious tissue changes and severe inflammation were spread out to places distant from the cauterized pancreas. In the film-treated group in particular, the accumulation of mono nuclear inflammatory cells was spread along the film, even into the outer zone of the sheet, and granulation formation of a barrier wall was suppressed around the film. Therefore, in the film-treated group, tissue injury and inflammation caused by pancreatic juice extended more widely and granulation formation of a barrier wall was strongly suppressed, even compared with that observed in the no treatment group. In conclusion, these fabrics can be a more suitable and effective material for the treatment of leakage of pancreatic juice.

手術中の制御困難な出血は死亡率増加や医療費の高額化をもたらすため、術中止血は重要な課題である。そのため、多くの局所止血材が開発されている。このうちで、タココンブ(TC)は最も使用頻度が高く効果的な局所止血材の一つである。しかし、TCの優れた止血効果はヒト血液製剤と動物組織由来の止血成分を構成要素として使用されていることによる。これはTCの使用により感染症やアレルギー反応のリスクが高くなる問題を引き起こす。この問題を解決するために、アルカリ処理によりゼラチンのウイルスや抗原性構造部位ほぼ完全に排除されることに着目し、筆者らは構成要素としてアルカリ処理ゼラチンのみを使用しこれを凍結乾燥してゼラチンスポンジ作成し、これとゼラチン糊とを組み合わせた新規の止血材を考案した。スポンジ構造が傷口からの流出血液を速やかに吸収してゼラチンと粘性ゲルを形成し、同時に血中凝固成分が直ちに活性化され、その結果、出血部位で急速に血餅形成が促進される。更にゼラチン糊が血餅を含む粘性ゲルを出血部位に強固に糊着させる。本実験では(A)ゼラチンスポンジとゼラチン糊との組み合わせ、(B)タココンブ単独、(C)ゼラチン糊単独の3種類の止血材について、犬の脾臓表面を損傷させた実験モデルを用いて止血効果を比較した。その結果、ゼラチンスポンジとゼラチン糊の併用は、他の2つの材料に比較して有意に優れた止血効果を示した。結論として、ゼラチンスポンジとゼラチン糊の組み合わせは、その優れた止血効果に加えて、感染症やアレルギー反応のリスクが極めて低い利点を持つ、有用な局所止血材であることが明らかになった。, Hemostasis is a key issue in surgery because uncontrolled surgical bleeding is associated with increased mortality rates and higher costs. Many types of topical hemostatic materials have been developed. One of the most current and most effective materials is TachoComb (TC), which is reabsorbed well by conversion into connective tissue. However, TC's excellent hemostatic effects are brought about by the use of hemostatic components and a framework derived from human and animal tissues. This means that TC use is associated with a risk of infectious diseases and allergic reactions. To address the above-mentioned problems with the most current hemostatic material, we devised a new hemostatic material composed of a framework of a freeze-dried gelatin sponge combined with gelatin glue, thereby creating a new topical hemostatic material made of gelatin alone. The gelatin used in the material was prepared with alkali-treatment, and the immunogenicity and activity of viruses were eliminated almost completely. The framework absorbs blood flowing out from the wound immediately, activates autologous coagulation components in the absorbed blood and promotes blood coagulation at the bleeding site. The hemostatic effects of three types of hemostatic materials were compared, namely (A) the gelatin sponge combined with gelatin glue, (B) TC and (C) gelatin glue alone, using a dog's spleen with a shaved wound surface. Consequently, combined use of the gelatin sponge and gelatin glue exhibited significantly superior hemostatic effects to the other two materials. In the conclusion, the combination of a gelatin sponge sheet and gelatin glue is useful as a topical hemostatic material due to its superior hemostatic effects and extremely low risk of infectious diseases and allergic reactions.

The diets of large, scarce mammals are difficult to study. We compared the food habits of Asiatic black bears (Ursus thibetanus) in the Tanzawa area, Japan, from before (1989–1993) and after (2001–2010) 2000 using both fecal contents and carbon and nitrogen stable isotope ratios in bear hairs. Bears ate the same foods before and after 2000, but some food categories differed in their frequencies of occurrence in autumn. Fruits of Prunus grayama, vine fruits, colonial insects, and Sasa vegetation declined and fruits of Swida controversa and sika deer (Cervus nippon) increased significantly as parts of the fall diet after 2000. Hair samples from 18 bears collected between 1973 and 2009 indicated that bears had higher summertime d 15 N rates after 2000 than before, indicating more meat in the diet. Thus, the Tanzawa bear population’s diet changed over 2 decades. Habitat conditions may have changed, causing bears to adjust their food habits. We discuss some factors, such as vegetation changes caused by overabundant deer or bears eating deer carcasses, that may be partly responsible. We argue that longterm diet studies are valuable for detecting habitat changes at scales that may not be apparent solely with habitat monitoring.

To achieve successful delivery of siRNA therapeutics, cytocleavable cationic polyrotaxanes (PRXs) composed of N,N-dimethylaminoethyl (DMAE) group-modified α-cyclodextrins (CDs) that were threaded onto a poly(ethylene glycol) (PEG) axis and capped with a bulky stopper using cytocleavable disulfide linkages (DMAE-PRX) were utilized as an siRNA carrier. DMAE-PRXs with various numbers of threading CDs and modified DMAE groups were synthesized, and the physicochemical properties, cellular internalization, and gene silencing activity of DMAE-PRX/siRNA were investigated to elucidate the relationship between its supramolecular structure and its function. When the numbers of modified DMAE groups were increased, the DMAE-PRXs formed closely associated polyplexes with siRNA and increased their polyanion exchange resistance. Additionally, the DMAE-PRXs with 52 threading CDs (52CD-PRXs) showed greater binding capabilities with siRNA and greater resistance to polyanion competition than 31CD-PRXs, indicating that the highly CD-threaded PRX structure in the 52CD-PRXs is superior in forming stable polyplexes with siRNA. Indeed, 52CD-PRX/siRNA showed greater intracellular uptake of siRNA than 31CD-PRX/siRNA with comparable numbers of DMAE groups. 52CD-PRX/siRNA successfully induced gene silencing of a targeted luciferase expressed in human cervical carcinoma without marked cytotoxicity and non-specific gene silencing. Although the gene silencing activities of DMAE-PRX/siRNA were comparable to those of linear poly(ethylenimine) (L-PEI), L-PEI showed cytotoxicity and non-specific gene silencing. Additionally, DMAE-PRXs with cytocleavable capabilities were found to enhance gene silencing, in comparison with non-cleavable DMAE-PRX. Thus, the cytocleavable cationic PRXs are suggested to be attractive supermolecules for the delivery of therapeutic siRNAs.

Background Ischemic mitral regurgitation (IMR) with ischemic cardiomyopathy (ICM) was treated with surgical procedures, and mitral leaflet tethering was assessed. Twenty-two patients with both ICM (left ventricular ejection fraction 2) underwent coronary artery bypass grafting (CABG), mitral annuloplasty (MAP) with or without surgical ventricular restoration (SVR) and procedures targeting the subvalvular apparatus. Methods and results Fourteen patients (group 1) underwent CABG and MAP, and the remaining 8 (group 2) underwent CABG, MAP, SVR, papillary muscle approximation (PMA), and papillary muscle suspension (PMS). PMA joined the entire papillary muscles with 3 mattress sutures. For PMS, 2 ePTFE sutures were placed between papillary muscle tips and fibrous annuli. Anterior and posterior mitral leaflet tethering angles (ALA and PLA) relative to the line connecting annuli, posterior and apical displacement of coaptation, and IMR grade were measured on echocardiography. Although preoperative ALA and PLA in group 2 were significantly larger than in group 1, there was no significant difference between groups at 1 month after surgery. At 1 year after surgery, however, the situation reversed: ALA and PLA in group 1 were significantly larger than in group 2. Conclusions In addition to MAP, procedures targeting the subvalvular apparatus including PMA and PMS achieved persistent reduction of mitral valve leaflet tethering, which might lead to the improvement of long-term outcome.

Multicellular neuronal activities should be investigated to reveal the dynamics of the neuronal circuit. Optical recording from neuronal populations is suitable for recording multicellular activities. We fabricated the prototype of the micro-imaging probe in combination with a gradient index lens and image fiber. This probe has a smaller diameter than traditional probes. We found an optimal optical configuration for maximizing the efficiency of the imaging probe. Using this optical configuration with the prototype of the imaging probe, the fluorescence images were captured from neurons expressing green fluorescent protein in a cerebellar block preparation, and the calcium-dependent images were sampled in a mouse brain slice preparation. Our optical system would facilitate the in vivo imaging studies with less invasive manners using thinner optic fiber than previously made.

Bone reconstruction is a challenging issue in the regeneration of surgically removed bone and disease-related bone defects. Although bone morphogenetic protein-2 (BMP-2) has received considerable attention as a bone regeneration inducer, a high dose of BMP-2 is typically required due to its short life-time under in vivo conditions. We have proposed a method to enhance the osteogenetic differentiation ability of BMP-2 in vitro that is based on supramolecular polyelectrolyte complexation with sulfonated polyrotaxanes (PRXs) consisting of sulfopropyl ether (SPE)-modified α-cyclodextrins threaded along a poly(ethylene glycol) chain capped with terminal bulky stopper molecules. In this study, we evaluated the in vivo bone regeneration ability of the SPE-PRX/BMP-2 complexes in a mouse calvarial defect model in comparison to free BMP-2 and heparin/BMP-2 complexes. The regenerated bone area was determined by X-ray computed microtomography, and the mice implanted with sulfonated PRX/BMP-2 complexes exhibited rapid and significant bone regeneration compared to those implanted with free BMP-2 and heparin/BMP-2 complexes. We concluded that the sulfonated PRX/BMP-2 complexes are a promising candidate for clinical bone regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1355-1363, 2017.

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.

Polyrotaxanes (PRXs) composed of threading methylated cyclodextrins exhibit a temperature-dependent reversible phase transition across the lower critical solution temperature (LCST) in water. In this study, a variety of methylated β-cyclodextrin (β-CD)-threaded PRXs (Me-PRXs) with different number of methyl groups modified on the β-CD and with different compositions of axle polymers (i.e., Pluronic, PEG-b-PPG-b-PEG) were synthesized, and the relationship between the LCST value and structural parameters was investigated. The LCST values of the Me-PRXs decreased with an increasing the methylation degree of the threading β-CD. Differential scanning calorimetric measurements demonstrated that the temperature-induced phase transition of the Me-PRXs occurred due to the dehydration of the threading methylated β-CD moieties and the subsequent hydrophobic interactions. Interestingly, the Me-PRXs formed coacervate droplets above their LCST. Altogether, we concluded that the LCST values of the Me-PRXs varied accordi...

Atopic dermatitis (AD) is a chronic inflammatory skin disease in humans. It was recently noted that the characteristics of epidermal barrier functions critically influence the pathological features of AD. Evidence suggests that claudin-1 (CLDN1), a major component of tight junctions (TJs) in the epidermis, plays a key role in human AD, but the mechanism underlying this role is poorly understood. One of the main challenges in studying CLDN1's effects is that Cldn1 knock-out mice cannot survive beyond 1 d after birth, due to lethal dehydration. Here, we established a series of mouse lines that express Cldn1 at various levels and used these mice to study Cldn1's effects in vivo. Notably, we discovered a dose-dependent effect of Cldn1's expression in orchestrating features of AD. In our experimental model, epithelial barrier functions and morphological changes in the skin varied exponentially with the decrease in Cldn1 expression level. At low Cldn1 expression levels, mice exhibited morphological features of AD and an innate immune response that included neutrophil and macrophage recruitment to the skin. These phenotypes were especially apparent in the infant stages and lessened as the mice became adults, depending on the expression level of Cldn1 Still, these adult mice with improved phenotypes showed an enhanced hapten-induced contact hypersensitivity response compared with WT mice. Furthermore, we revealed a relationship between macrophage recruitment and CLDN1 levels in human AD patients. Our findings collectively suggest that CLDN1 regulates the pathogenesis, severity, and natural course of human AD.

Photobiomodulation (PBM) is a noninvasive treatment that can be neuroprotective, although the underlying mechanisms remain unclear. In the present study, we assessed the mechanism of PBM as a novel treatment for noise-induced hearing loss, focusing on the nuclear factor (NF)-κB signaling pathway. Sprague-Dawley rats were exposed to 1-octave band noise centered at 4kHz for 5h (121dB). After noise exposure, their right ears were irradiated with an 808nm diode laser beam at an output power density of 165mW/cm(2) for 30min a day for 5 consecutive days. Measurement of the auditory brainstem response revealed an accelerated recovery of auditory function in the groups treated with PBM compared with the non-treatment group at 4, 7, and 14 days after noise exposure. Immunofluorescent image analysis for inducible nitric oxide synthase and cleaved caspase-3 showed lesser immunoreactivities in outer hair cells in the PBM group compared with the non-treatment group. However, immunofluorescent image analysis for NF-κB, an upstream protein of inducible nitric oxide synthase, revealed greater activation in the PBM group compared with the naïve and non-treatment groups. Western blot analysis for NF-κB also showed stronger activation in the cochlear tissues in the PBM group compared with the naïve and non-treatment groups (p0.01, each). These data suggest that PBM activates NF-κB to induce protection against inducible nitric oxide synthase-triggered oxidative stress and caspase-3-mediated apoptosis that occur following noise-induced hearing loss.

Claudin protein family members, of which there are at least 27 in humans and mice, polymerize to form tight junctions (TJs) between epithelial cells, in a tissue- and developmental stage-specific manner. Claudins have a paracellular barrier function. In addition, certain claudins function as paracellular channels for small ions and/or solutes by forming selective pores at the TJs, although the specific claudins involved and their functional mechanisms are still in question. Here we show for the first time that claudin-21, which is more highly expressed in the embryonic than the postnatal stages, acts as a paracellular channel for small cations, such as Na(+), similar to the typical channel-type claudins claudin-2 and -15. Claudin-21 also allows the paracellular passage of larger solutes. Our findings suggest that claudin-21-based TJs allow the passage of small and larger solutes by both paracellular channel-based and some additional mechanisms.

Helicobacter cinaediis 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 withHelicobacter cinaedi, treated successfully with surgical repair and long-term antimicrobial therapy.

Tight junctions (TJs) connect epithelial cells and form a semipermeable barrier that only allows selective passage of ions and solutes across epithelia. Here we show that mice lacking EpCAM, a putative cell adhesion protein frequently overexpressed in human cancers, manifest intestinal barrier defects and die shortly after birth as a result of intestinal erosion. EpCAM was found to be highly expressed in the developing intestinal epithelium of wild-type mice and to localize to cell–cell junctions including TJs. Claudin-7 colocalized with EpCAM at cell–cell junctions, and the two proteins were found to associate with each other. Claudins 2, 3, 7, and 15 were down-regulated in the intestine of EpCAM mutant mice, with claudin-7 being reduced to undetectable levels. TJs in the mutant intestinal epithelium were morphologically abnormal with the network of TJ strands scattered and dispersed. Finally, the barrier function of the intestinal epithelium was impaired in the mutant animals. These results suggest that EpCAM contributes to formation of intestinal barrier by recruiting claudins to cell–cell junctions.

コラーゲンやゼラチンのアルカリ抽出法は、従来の酸処理や酵素処理法に比べ幾つかの優れた利点を有する。一方で、その過程において等電点が酸性化する事が知られており、その生物学的な効果や影響については未だ十分に明らかになっていない。そこで本論文では、そのようなアルカリ処理コラーゲンやゼラチンの生物学的性質を調べるため、足場材料として用いた場合の細胞増殖に対する影響について予備的に検討を行った。さらにアルカリ処理コラーゲンやゼラチンに対する架橋修飾による影響、および反対の等電点をもつコラーゲンやゼラチンとの混合による影響等についても検討を行った。ペプシン処理コラーゲン、アルカリ処理コラーゲン、酸処理ゼラチン、アルカリ処理ゼラチンを、それぞれコーティングしたプレート上においてラット培養細胞を培養し、4日後その増殖の程度について評価を行った。結果、アルカリ処理コラーゲンやゼラチンにおいて、細胞増殖の低下が認められた。しかしながらUV架橋を行ったところ、その細胞増殖が回復することが認められた。また、ペプシン処理コラーゲンとアルカリ処理コラーゲンを混合した場合、少量を混合した場合は特に影響を認めなかったが、等量近く混合した場合においては、細胞増殖が極端に低化していた。一方酸処理ゼラチンとアルカリ処理ゼラチンを混合した場合においても、細胞増殖の低下は認められたが、その極端な低下は認められなかった。以上より、アルカリ処理コラーゲンやゼラチンを足場として用いた場合、細胞の増殖はむしろ低下していた。しかしながら、適度の架橋を加える、あるいは従来のアルカリ性の等電点を持つコラーゲンやゼラチンと適切な配合比において混合使用することにより、足場機能を損なわずに、そのアルカリ処理による利点を生かした有用な足場材料として使用できる可能性があると考えられた。, The alkaline treatment to extract collagen or gelatin has several benefits, compared with conventional acid or enzymatic treatment. However, during such alkaline processing, the isoelectric points of collagen and gelatin are shifted from alkalinity to acidity, and the biological influences have not yet been fully established. In order to clarify the biological properties of the alkali-treated collagen or gelatin, we performed a preliminary examination of the influence on the cell growth when these materials are used as scaffolds. Furthermore, we studied the effects of the alkali-treated collagen and gelatin on cell growth by UV cross-linking or by mixing them with the conventional pepsin-treated collagen or acid-treated gelatin showing the opposite isoelectric points. After four days in culture on the plates coated with pepsin-processed collagen (pep-collagen), alkali-treated collagen (alk-collagen), acid-treated gelatin (acd-gelatin) or alkali-treated gelatin (alk-gelatin), the number of rat fibroblasts was evaluated. The cell growth on the alk-collagen and gelatin decreased, in comparison with the control (non-coated). However, the cell number on the alk-collagen and -gelatin was improved when they were UV cross-linked. The cell growth was not affected when a small amount of pep- or alk-collagen was mixed with alk- or pep-collagen, respectively. However, the cell growth was decreased remarkably, when these collagens were mixed at a nearly equal ratio. The inhibition of the cell growth was also seen after mixing acd-gelatin and alk-gelatin. However, this inhibition of the cell growth was not significant. In summary, the alk-collagen and gelatin decreased the cell growth rather than control when used as scaffolds. However, by adding UV cross-linking or by mixing them with conventional collagen or gelatin at suitable compounding ratios, these materials may be therefore utilized as a more useful scaffold without spoiling their function.

Summary Background Some cases of human papillomavirus (HPV) type 56 infection in Bowen disease have been reported. However, the incidence and clinical characteristics are still unclear. Objective To clarify the prevalence of HPV type 56-positive Bowen disease in our department and to characterize the clinical manifestations. Methods Sixty-eight specimens of Bowen disease were examined by polymerase chain reaction using HPV consensus primers, and the amplified products were subjected to DNA sequence analyses. Moreover, positive samples were investigated by in situ hybridization. These findings were used to clarify the clinical characteristics of HPV-positive Bowen disease. Results Eight out of 68 specimens (12%) of Bowen disease were HPV-positive, of which six specimens were HPV type 56-positive. The HPV type 56-positive lesions were characterized by a longitudinal melanonychia or a deeply pigmented keratotic lesion. The remaining two specimens were genital Bowen disease in which HPV type 16 was detected. In situ hybridization demonstrated the positive cells in the upper layer of epidermis. The HPV type 56 detected in the samples of longitudinal melanonychia can be divided into at least into two types. Conclusions This study determined the prevalence of HPV type 56-positive Bowen disease. Longitudinal melanonychia is the most characteristic manifestation of HPV type 56-associated Bowen disease.

The claudins comprise a multigene family that consists of at least 27 members. Claudins are responsible for establishing the paracellular barrier--which has permselectivity--at the tight junctions in epithelial cells, and the specific patterns of claudin expression in the epithelial cell sheets that cover the internal and external surfaces of organs contribute to the formation of microenvironments and organs' biological functions. Data on the detailed characterization of individual claudins and their roles in different microenvironments are accumulating. A study on the stomach-specific claudin-18-knockout mouse, which has gastritis, recently revealed that the stomach-type claudin-18 specifically forms the proton barrier in the stomach, consistent with previously reported circumstantial evidence. Combined with previous studies on the specific ionic homeostasis by different types of claudins, our findings support the idea that claudins may regulate ion-specific homeostasis in vivo.

Cell cultivation on the surface of microcarriers in stirred suspension is an essential method for the large-scale culture of anchorage-dependent cells. For applying this method to the field of cell therapy and for obtaining a large number of intact cells, non-invasive cell harvest without proteolytic enzyme treatment is an advantageous method. In this regard, temperature-responsive microcarriers that bearing poly(N-isopropylacrylamide) (PIPAAm)-grafted chains on the outermost surface were developed for harvesting cultured cells by temperature alteration. PIPAAm-grafted beads with the various grafted amount of PIPAAm and various bead diameters were synthesized for optimizing cell proliferation and thermally-induced detachment on the surface. The chinese hamster ovary (CHO-K1) cells adhered on the surface of all PIPAAm-grafted beads at 37 °C, while the adhering cells were found to detach themselves from the surfaces at 20 °C. The efficiency of thermally-induced cell detachment increased with increasing the grafted amount of PIPAAm and the diameter of bead. An efficient cell proliferation on bead surfaces in stirred suspension culture and subsequent thermally-induced cell detachment were achieved by the precise regulation of both the grafted amount of PIPAAm and diameter of bead. The temperature-responsive microcarriers exhibiting temperature-dependent cell adhesion and detachment will be an attractive candidate for the large-scale cell culture of therapeutic cells.

Objectives: Early and mid-term results of stent graft (SG) treatment for thoracic aortic aneurysms (thoracic endovascular aneurysm repair: TEVAR) were retrospectively compared with open surgical treatment. Methods: The records of 213 patients in whom single thoracic aortic aneurysm repairs had been performed in our department from January 2006 through August 31, 2009 were reviewed. Acute aortic dissection was excluded. Each case was reviewed for indications for TEVAR from an anatomical standpoint. Among 62 cases in which TEVAR was indicated, 30 (SG group) were treated by TEVAR and 32, by open surgery (OP group). Early and mid-term results were analyzed retrospectively in both groups. Results: There were no operative deaths in either group. The SG group demonstrated significantly less operative bleeding, a shorter operative time, and shorter postoperative hospital stay compared with the OP group. There were 3 deaths in the SG group and 4 in the OP group, which occurred within an average of 656.4 days during the follow up period. The 3 year actuarial survival rate was 88.7% in the SG group and 87.1% in the OP group, and there were no significant differences between the groups. Conclusion: Although early and mid-term results of TEVAR and open surgery were similar, TEVAR is generally less invasive and may be preferable for high-risk patients, compared with open surgical repair. (English Translation of Jpn J Vasc Surg 2010; 19: 51-56.)

Objective: This paper aims to comprehensively document a rare case of sensorineural hearing loss accompanied by hemophagocytic syndrome which could be one of the causes of cochlear dysfunction. Methods: A 63-year-old man presented with right-sided sudden hearing loss and dizziness and pure-tone audiometry showed a right-sided sensorineural hearing loss. The patient was treated with steroid pulse infusion therapy, ATP and vitamin B12. Results: Bone marrow specimen revealed hemophagocytic cells and the diagnosis of hemophagocytic syndrome was made. A blood culture grew Capnocytophaga. The prognosis for hearing recovery was poor. Conclusion: Although viral infection and cochlear circulatory dysfunction have been suggested to be associated with sudden sensorineural hearing loss, hemophagocytic syndrome due to bacterial infection which produces high cytokine levels might cause dysfunction of the inner ear.

Tricellulin (also known as MARVELD2) is considered as a central component of tricellular tight junctions and is distributed among various epithelial tissues. Although mutations in the gene encoding tricellulin are known to cause deafness in humans (DFNB49) and mice, the influence of its systemic deletion in vivo remains unknown. When we generated tricellulin-knockout mice (Tric−/−), we found an early-onset rapidly progressive hearing loss associated with the degeneration of hair cells (HCs); however, their body size and overall appearance were normal. Tric−/− mice did not show any morphological change pertaining to other organs such as the gastrointestinal tract, liver, kidney, thyroid gland and heart. The endocochlear potential (EP) was normal in Tric−/− mice, suggesting that the tight junction barrier is maintained in the stria vascularis, where EP is generated. The degeneration of HCs, which occurred after the maturation of EP, was prevented in the culture medium with an ion concentration similar to that of the perilymph. These data demonstrate the specific requirement of tricellulin for maintaining ion homeostasis around cochlear HCs to ensure their survival. The Tric−/− mouse provides a new model for understanding the distinct roles of tricellulin in different epithelial systems as well as in the pathogenesis of DFNB49.

We report pH-responsive structural changes in PEG-modified (PEGylated) nanogels, as determined by using small-angle X-ray scattering and dynamic light scattering. The size of the nanogels discontinuously increased at a lower pH than the pKa of the nanogels. This size increase was attributed to the swelling of the core part of the nanogel upon a change in pH. The swelling behavior was dependent on the cross-link density of the core. When the cross-link density was low, the core swelled greatly with preserving the polydispersity in size and maintained a constant shape; however, when the cross-link density was high, the core swelled minimally, and only the polydispersity increased. This difference in swelling behavior is discussed in terms of the inhomogeneous structural distribution of cross-links in the core. By using small-angle X-ray scattering and dynamic light scattering, pH-responsive swelling behavior of the core part of PEGylated nanogels was investigated. The swelling behavior depended on the cross-link density of the core part. When the cross-link density was small, the core swelled with keeping the polydispersity in size and shape constant; to the contrary, the core hardly swelled and only the polydispersity increased when the cross-link density was large. This difference in the swelling behavior is discussed in terms of inhomogeneous structural distribution of cross-links in the core.

The biocompatibility and body distribution of PEGylated polyamine nanogels composed of chemically cross-linked poly(2-N,N-(diethylamino)ethyl methacrylate) (PEAMA) gel cores surrounded by poly(ethylene glycol) (PEG) chains were investigated to evaluate their feasibility as drug nanocarriers for systemic administration. PEGylated nanogels with different cross-linking densities (1, 2, and 5 mol.%) were prepared to evaluate their biocompatibilities by in vitro cytotoxicity assay, hemolysis assay, and in vivo acute toxicity assay. The toxic effect of the PEGylated nanogels derived from polyamine gel cores was significantly reduced when the cross-linking density was increased, and those with a cross-linking density of 5 mol.% showed a remarkably high median lethal dose (LD50) value >200 mg kg−1,despite the abundance of amino groups in the core. One hour after intravenous injection the PEGylated nanogels were found to have been eliminated from the systemic circulation, and less than 1% of the injected dose (ID) remained in the bloodstream. To improve the blood circulation time by increasing the surface PEG density of the PEGylated nanogels post-PEGylation of the PEGylated nanogels (via the Menschutkin reaction between tertiary amines of the PEAMA gel core and bromobenzyl-terminated short PEG) was carried out. A biodistribution study of these post-PEGylated nanogels revealed that the blood circulation time of the nanogels was definitely prolonged as the PEG content was increased. Therefore, the precise design of PEGylated nanogels with increased cross-linking densities in their polyamine gel cores and increased surface PEG densities seems promising for systemic applications.

Background & Aims In the small intestine, the paracellular transport of Na + is thought to be critical for luminal Na + -homeostasis and the transcellular absorption of nutrients by Na + -driven transporters. Na + is supplied to the intestinal lumen from the submucosa and serum through tight junctions, which form a paracellular barrier between the cells of epithelial sheets. However, the molecular basis for this paracellular transport of Na + is not well understood. Here, we examined this mechanism by performing loss-of-function studies of claudin-2 and claudin-15, two tight-junctional membrane proteins that are specifically and age-dependently expressed in the villi and/or crypts of small intestinal epithelia. Methods Knockout mice for claudin-2 or claudin-15 were subjected to histologic, cell biologic, electrophysiologic, and physiologic analyses. Results Examination of the knockout mice revealed that both claudin-2 and claudin-15 play crucial roles in the transepithelial paracellular channel-like permselectivity for extracellular monovalent cations, particularly Na + , in infants and adults. Especially in Cldn15 −/− adults, the luminal Na + concentration in the small intestine measured directly in vivo was abnormally low, and glucose absorption was impaired, as assessed by the oral glucose tolerance test and estimation of unabsorbed glucose. Conclusions We propose that the "Na + -leaky" claudin-15 is indispensable in vivo for the paracellular Na + permeability, luminal Na + -homeostasis, and efficient glucose absorption in the small intestine, but claudin-2 is indispensable for only the first of these functions. Claudin-15 knockout leads to Na + deficiency and glucose malabsorption in the mouse adult small intestine.

Claudins (Cldn) are essential membrane proteins of tight junctions (TJs), which form the paracellular permselective barrier. They are produced by a multi-gene family of 24 reported members in mouse and human. Based on a comprehensive search combined with phylogenetic analyses, we identified three novel claudins (claudin-25, -26, and -27). Quantitative RT-PCR revealed that the three novel claudins were expressed in a tissue- and/or developmental stage-dependent manner. Claudins-25 and -26, but not claudin-27, were immunofluorescently localized to TJs when exogenously expressed in cultured MDCK and Eph epithelial cell lines. These findings expand the claudin family to include at least 27 members.Structured summaryClaudin-25 and ZO-1 colocalize by fluorescence microscopy (View interaction)ZO-1 and Claudin-26 colocalize by fluorescence microscopy (View interaction)

Tight junctions are the main intercellular junctions of podocytes of the renal glomerulus under nephrotic conditions. Their requisite components, claudins, still remain to be identified. We have measured the mRNA levels of claudin subtypes by quantitative real-time PCR using isolated rat glomeruli. Claudin-5 was found to be expressed most abundantly in glomeruli. Mass spectrometric analysis of membrane preparation from isolated glomeruli also confirmed only claudin-5 expression without any detection of other claudin subtypes. In situ hybridization and immunolocalization studies revealed that claudin-5 was localized mainly in glomeruli where podocytes were the only cells expressing claudin-5. Claudin-5 protein was observed on the entire surface of podocytes including apical and basal domains of the plasma membrane in the normal condition and was inclined to be concentrated on tight junctions in puromycin aminonucleoside nephrosis. Total protein levels of claudin-5 in isolated glomeruli were not significantly upregulated in the nephrosis. These findings suggest that claudin-5 is a main claudin expressed in podocytes and that the formation of tight junctions in the nephrosis may be due to local recruitment of claudin-5 rather than due to total upregulation of the claudin protein levels.