Generation of retinal pigment epithelium from human induced pluripotent stem cells showed polarized secretion of VEGF and PEDF.

Age-related macular degeneration (AMD) is one of the major diseases that cause severe visual impairment in developed countries and is related to dysfunction of the retinal pigment epithelium (RPE). Human induced pluripotent stem cells (hiPSCs) have been drawing attention as a valuable source of RPE for basic research on or treatment of AMD. Here we investigated whether RPE could be generated from hiPSCs in Kawasaki Medical School. We maintained hiPSCs retaining pluripotent markers after a series of regular culture steps involving passaging, freezing, and thawing. hiPSCs were then cultured in RPE differentiation medium, and pigmented colonies were manually isolated for further differentiation into RPE. Differentiated cells formed pigmented cells with a typical RPE cobblestone appearance, abundant apical microvilli, adherens junctions, and tight junctions. Furthermore, generated pigmented cells expressed typical RPE marker genes, exhibited a barrier function, and secreted growth factors in a polarity-dependent manner similar to native RPE. These results indicate that RPE derived from hiPSCs in our facility can be used for in vitro and in vivo research

Cultivable Anaerobic Microbiota of Infected Root Canals

Objective. Periapical periodontitis is an infectious and inflammatory disease of the periapical tissues caused by oral bacteria invading the root canal. In the present study, profiling of the microbiota in infected root canals was performed using anaerobic culture and molecular biological techniques for bacterial identification. Methods. Informed consent was obtained from all subjects (age ranges, 34–71 years). Nine infected root canals with periapical lesions from 7 subjects were included. Samples from infected root canals were collected, followed by anaerobic culture on CDC blood agar plates. After 7 days, colony forming units (CFU) were counted and isolated bacteria were identified by 16S rRNA gene sequencing. Results. The mean bacterial count (CFU) in root canals was (0.5 ± 1.1) × 106 (range 8.0 × 101–3.1 × 106), and anaerobic bacteria were predominant (89.8%). The predominant isolates were Olsenella (25.4%), Mogibacterium (17.7%), Pseudoramibacter (17.7%), Propionibacterium (11.9%) and Parvimonas (5.9%). Conclusion. The combination of anaerobic culture and molecular biological techniques makes it possible to analyze rapidly the microbiota in infected root canals. The overwhelming majority of the isolates from infected root canals were found to be anaerobic bacteria, suggesting that the environment in root canals is anaerobic and therefore support the growth of anaerobes

Rapid Quantification of Bacteria in Infected Root Canals Using Fluorescence Reagents and a Membrane Filter: A Pilot Study on Its Clinical Application to the Evaluation of the Outcomes of Endodontic Treatment

Objective. The bacterial examination has been performed during the course of the root canal treatment. In the present pilot study, the new developed method, using fluorescence reagents and a membrane filter, was applied to the detection and quantification of bacteria in infected root canals, in order to evaluate the outcomes of the treatment. Methods. Six infected root canals with periapical lesions from 5 subjects were included. Informed consent was obtained from all subjects (age ranges, 23–79 years). Samples from infected root canals were collected at the beginning of the treatment (termed #25 First), the end of the first day of treatment (termed #55 First), and the next appointment day (termed #55 Second). Then, the bacterial count (CFU) was measured using fluorescence reagents (4′,6′-diamidino-2-phenylindole and propidium iodide) and the polycarbonate membrane filter by Bioplorer. Results. The mean ± SD of CFU in the sample of “#25 First” was (1.0 ± 1.4) × 105. As the root canal treatment progressed, the CFU decreased as 7.9 × 103 (#55 First) and 4.3 × 102 (#55 Second). Conclusion. In the present pilot study, rapid detection and quantification of bacteria in infected root canals were found to be successfully performed using fluorescence reagents and a membrane filter (Bioplorer analysis)

給食施設における献立作成業務実態調査 : 作業の所要時間と標準化にむけて

献立は食事の設計品質を示したものであり、 作成には高度な専門知識と技術を要する。 献立を標準化する上で、 栄養管理業務の献立作成作業時間に着目し、 実務における献立作成業務の状況と管理栄養士・栄養士が抱えている課題を調査し、 作業時間を要する因子を検討した。 献立作成の作業時間は、 管理栄養士8.0±6.5時間/週、 栄養士は 7.7 ±6.6時間/週であった。 献立作成の主観的困難さの理由として、 個人の嗜好やアレルギーなどで献立に使用できない食品数 (禁止食品) に関する事例が多くあげられたが、 禁止食品と作業時間の関連がみられなかった。 業務の複雑さとの関連をさらに精査し、 給食経営作業の標準化について検討する必要が示唆された。Targeting "dieticians" and "national registered dieticians", we conducted a questionnaire to investigate whether there is a difference in the time required for menu planning caused by the difference in the type of license. In addition, we clarify the problems in menu planning. The hours worked for menu planning were 8.0±6.5 per week for national registered dieticians, and 7.7±6.6 per week for dieticians. Regarding the problems related to menu planning, a number of cases were reported with respect to the number of food items that cannot be included in the menu because of personal preferences or allergies. A study on the standardization of menu planning operations is required

iPS 細胞由来網膜色素上皮細胞移植のためのNaIO3誘因網膜色素上皮変性げっ歯類モデルの特性評価

網膜色素上皮（RPE）は視細胞維持に重要な役割を担い，その機能低下は加齢黄斑変性等の原因となる．近年，健常なRPE を補充するRPE 移植が注目されており，臨床応用も開始された．前臨床研究における動物実験にあたり，過去にもげっ歯類のヨウ素酸ナトリウム（NaIO3）誘因RPE 変性モデルの研究はあるが，げっ歯類は黄斑部を欠くため最適でない．黄斑を有する非ヒト霊長類における薬剤全身投与は両眼の失明を招くため動物愛護の観点から不適切であり，現在適切なモデルが存在しない．従って，我々はサルを用いた移植実験を念頭に置きNaIO3の硝子体内投与によりラット片眼にRPE 変性を誘発可能か研究した．また，NaIO3誘因RPE 変性モデルにヒト多能性幹細胞由来RPE（hiPS-RPE）を網膜下移植し視細胞の保護効果を検討した．8-10週齢のオスのWistar ラットの片眼に0.005，0.01，0.02 mg/μl のNaIO3を硝子体内投与した．経時的にHE 染色にて網膜外層厚と網膜内層厚を計測し，透過型電子顕微鏡で形態学的評価を行った．NaIO3投与後2日目にhiPS-RPE 懸濁液を網膜下に移植し，組織学的評価を行った．0.005 mg/μl 投与群では網膜内層・外層厚ともに変化はなく，0.02 mg/μl 投与群では網膜全層が障害され，0.01 mg/μl 投与群では網膜外層のみが障害された．RPE の形態学的変化はNaIO3投与後3日目から出現し，以降網膜外層厚が急速に菲薄化した．いずれの投与群も非投与眼に影響はなかった．hiPS-RPE 移植眼では，網膜外層厚が厚い傾向にあった．まとめると，NaIO3片眼硝子体内投与により，げっ歯類片眼RPE 障害モデル作製に成功した．hiPS-RPE 網膜下移植により視細胞保護効果を得られた．本方法はRPE 移植の前臨床研究の動物モデルの基礎として有用である．The retinal pigment epithelium (RPE) plays an important role in maintaining photoreceptors, and RPE dysfunction causes eye diseases such as age-related macular degeneration (AMD). RPE transplantation has garnered much attention in recent years as a way to replenish healthy RPE, and clinical applications for RPE transplantation in patients with AMD have been reported. Preclinical studies use animal models to examine treatment efficacy, however, whether conventional methods with animal models are suitable for examining RPE transplantation is controversial. Systemic administration of sodium iodate (NaIO3) has beenshown to induce RPE degeneration in rodent models, recapitulating morphological atrophy in corresponding areas. However, as rodents lack a macular region, these studies cannot precisely evaluate the effects of systemic administration. The induction of blindness in both eyes in nonhuman primates bearing a macular region is also inappropriate in terms of animal ethics. With this in mind, we investigated whether intravitreal injection of NaIO3 can selectively induce a single-eye impairment in an RPE animal model. Additionally, we investigated whether subretinal transplantation of human induced pluripotent stem cells-derived RPE (hiPS-RPE) can protect against NaIO3-induced RPE degeneration.The intravitreal injection of NaIO3 was performed in 8-10 week-old male Wistar rats with rats being grouped by dose (0.005, 0.01, and 0.02 mg/μl). We measured outer nuclear layer (ONL) thickness and inner nuclear layer (INL) thickness at 8 retinal points by hematoxylin-eosin staining and evaluated RPE morphology by transmission electron microscopy sequentially (3, 7, 14, and 28 days after injection). In addition, hiPSC-RPE cell suspension was injected into the subretinal space 2 days after NaIO3 administration and histological evaluation was performed sequentially (7, 14, and 28 days after NaIO3 injection).The results showed no significant changes in INL and ONL thicknesses in the 0.005 mg/μl group. Retinal thicknesses in the 0.02 mg/μl group had almost completely degenerated. In the 0.01 mg/μl group, ONL thickness had almost completely degenerated, but, in contrast INL thickness was preserved as well as 0.005 mg/μl injection group. RPE morphological changes and TUNEL-positive cells appeared at 3 days after NaIO3 injection, followed by rapid thinning of ONL thickness. None of the retinal thickness of contralateral eye in all groups showed any changes. Additionally, ONL thicknesses in the transplanted eyes were significantly greater than those in the nontransplanted and sham-surgery groups

高校生における異文化体験と国際的資質の関連 : 海外研修旅行の効果

本研究の目的は， 日本の高校生を対象に，国際的資質が海外研修旅行における異文化体験へ与える影響を検討することであった。3か国のうち1か国を選択して海外研修旅行を経験した高校生158人に対し，国際的資質および異文化体験を測定する賀問紙調査を実施した。その結呆，旅行後の国際的資質が有意に高まったが，その効果量は低かった。また異文化に対する認識の肯定的変化は，旅行の結果高まったほか，旅行前の国際的資質に強く影響受けることが明らかとなった。教育効果の検証における個人要因導入の必要性について考察された。The purpose of this study was lo examine the impact of international disposition on cross-cultural experiences in an oversea school trip among Japanese high school students. 158 high school students who participated in one in three foreign countries\u27 trip answered the questionnaire regarding international disposition and cross-cultural experiences. The results showed that scores of international disposition significantly increased but the effect sizes were moderate. Positively changes of the realization toward other countries also increased and were positively influenced by international disposition before the trip. Application of individual factors to the examination of the teaching effectiveness was discussed

Transcriptome Analysis of the Hierarchical Response of Histone Deacetylase Proteins That Respond in an Antagonistic Manner to Salinity Stress

Acetylation in histone and non-histone proteins is balanced by histone acetyltransferase and histone deacetylase (HDAC) enzymatic activity, an essential aspect of fine-tuning plant response to environmental stresses. HDACs in Arabidopsis are composed of three families (RPD3-like, SIRT, and HD-tuins). A previous study indicated that class I (HDA19) and class II (HDA5/14/15/18) RPD3-like family HDACs control positive and negative responses to salinity stress, respectively. Furthermore, quintuple hda5/14/15/18/19 mutants (quint) exhibit salinity stress tolerance, suggesting that hda19 suppresses the sensitivity to salinity stress present in quadruple hda5/14/15/18 mutants (quad). In the present study, transcriptome analysis of the quint mutant was conducted to elucidate the hierarchical control of salinity stress response operated by RPD3-like family HDACs (HDA5/14/15/18/19). The analysis identified 4,832 salt-responsive genes in wild-type (Col-0), hda19-3, quad, and quint plants and revealed that 56.7% of the salt-responsive genes exhibited a similar expression pattern in both the hda19-3 and quint plants. These results indicate that deficiency in HDA19 has a bigger impact on salinity stress response than in class II HDACs. Furthermore, the expression pattern of genes encoding enzymes that metabolize phytohormones raises the possibility that a drastic change in the homeostasis of phytohormones, such as abscisic acid, brassinosteroid, and gibberellin, may contribute to increasing stress tolerance in hda19-3 and quint plants. Among these phytohormones, abscisic acid accumulation actually increased in hda19-3 and quint plants, and decreased in quad, compared with wild-type plants. Importantly, 7.8% of the salt-responsive genes in quint plants exhibited a similar expression pattern in quad plants, suggesting that some gene sets are regulated in an HDA5/14/15/18-dependent manner. The transcriptome analysis conducted in the present study revealed the hierarchical and independent regulation of salt stress response that is mediated through HDA19 and class II HDACs