Construction of the lactate-sensing fibremats by confining sensor fluorescent protein of lactate inside nanofibers of the poly(HPMA/DAMA)/ADH-nylon 6 core-shell fibremat.

The development of a new materials platform capable of sustaining the functionality of proteinous sensor molecules over an extended period without being affected by biological contaminants in living systems, such as proteases, is highly demanded. In this study, our primary focus was on fabricating new core-shell fibremats using unique polymer materials, capable of functionalizing encapsulated sensor proteins while resisting the effects of proteases. The core-fibre parts of core-shell fibremats were made using a newly developed post-crosslinkable water-soluble copolymer, poly(2-hydroxypropyl methacrylamide)-co-poly(diacetone methacrylamide), and the bifunctional crosslinking agent, adipic dihydrazide, while the shell layer of the nanofibers was made of nylon 6. Upon encapsulating the lactate-sensor protein eLACCO1.1 at the core-fibre part, the fibremat exhibited a distinct concentration-dependent fluorescence response, with a dynamic range of fluorescence alteration exceeding 1000% over the lactate concentration range of 0 to 100 mM. The estimated dissociation constant from the titration data was comparable to that estimated in a buffer solution. The response remained stable even after 5 cycles and in the presence of proteases. These results indicates that our core-shell fibremat platform could serve as effective immobilizing substrates for various sensor proteins, facilitating continuous and quantitative monitoring of various low-molecular-weight metabolites and catabolites in a variety of biological samples.

Core-shell fibremats comprising a poly(AM/DAAM)/ADH nanofibre core and nylon6 shell layer are an attractive immobilization platform for constructing immobilised enzymes.

Core-shell fibremats, comprising poly(acrylamide)-co-poly(diacetone-acrylamide)/adipic dihydrazide [poly(AM/DAAM)/ADH] core-nanofibres and hydrophobic polymer shell layers, are a new class of platforms for constructing various immobilised enzymes. In this study, to elucidate the impacts of the shell-layer material on fibremat properties and enzymatic activities, we synthesised core-shell fibremats with shell layers comprising nylon6 or acetyl cellulose (AcCel) instead of poly(ε-caprolactone) (PCL), as in our previous study. Transmission and scanning electron microscopy images revealed that the lactase-encapsulated poly(AM/DAAM)/ADH-nylon6 and -AcCel fibremats were both constructed like the poly(AM/DAAM)/ADH-PCL one. Leakage measurements of the beforehand loaded molecules inside the core-nanofibres revealed that both fibremats exhibited efficient permeability for low-molecular-weight molecules and stable retention of enzyme molecules inside the core-nanofibres. Meanwhile, the fibremats' mechanical properties considerably depended on the choice of shell-layer material. The thermal analyses of the lactase-encapsulated fibremats revealed residual water inside the core nanofibres. The core-shell fibremats fabricated with a nylon6 or PCL shell exhibited excellent enzymatic activities (102 and 114%, respectively, compared to that of free lactase), superior to that of the same amount of free enzyme in a buffer. Furthermore, both core-shell fibremats retained over 95% of their initial enzymatic activities, even after they were re-used 10 times.

Projection of Damaged Visual and Language Regions on Low Trail Making Test Part-B Performance in Stroke Patients.

Background: The Trail Making Test Part-B (TMT-B) is an attention functional test to investigate cognitive dysfunction. It requires the ability to recognize not only numbers but also letters. We analyzed the relationship between brain lesions in stroke patients and their TMT-B performance. Methods: From the TMT-B, two parameters (score and completion time) were obtained. The subjects were classified into several relevant groups by their scores and completion times through a data-driven analysis (k-means clustering). The score-classified groups were characterized by low (≤10), moderate (10 < score < 25), and high (25) scores. In terms of the completion time, the subjects were classified into four groups. The lesion degree in the brain was calculated for each of the 116 regions classified by automated anatomical labeling (AAL). For each group, brain sites with a significant difference (corrected p < 0.1) between each of the 116 regions were determined by a Wilcoxon Rank-Sum significant difference test. Results: Lesions at the cuneus and the superior occipital gyrus, which are mostly involved in visual processing, were significant (corrected p < 0.1) in the low-score group. Furthermore, the moderate-score group showed more-severe lesion degrees (corrected p < 0.05) in the regions responsible for the linguistic functions, such as the superior temporal gyrus and the supramarginal gyrus. As for the completion times, lesions in the calcarine, the cuneus, and related regions were significant (corrected p < 0.1) in the fastest group as compared to the slowest group. These regions are also involved in visual processing. Conclusion: The TMT-B results revealed that the subjects in the low-score group or the slowest- group mainly had damage in the visual area, whereas the subjects in the moderate-score group mainly had damage in the language area. These results suggest the potential utility of TMT-B performance in the lesion site.

Artificially-reconstructed brain images with stroke lesions from non-imaging data: modeling in categorized patients based on lesion occurrence and sparsity.

Brain imaging is necessary for understanding disease symptoms, including stroke. However, frequent imaging procedures encounter practical limitations. Estimating the brain information (e.g., lesions) without imaging sessions is beneficial for this scenario. Prospective estimating variables are non-imaging data collected from standard tests. Therefore, the current study aims to examine the variable feasibility for modelling lesion locations. Heterogeneous variables were employed in the multivariate logistic regression. Furthermore, patients were categorized (i.e., unsupervised clustering through k-means method) by the charasteristics of lesion occurrence (i.e., ratio between the lesioned and total regions) and sparsity (i.e., density measure of lesion occurrences across regions). Considering those charasteristics in models improved estimation performances. Lesions (116 regions in Automated Anatomical Labeling) were adequately predicted (sensitivity: 80.0-87.5% in median). We confirmed that the usability of models was extendable to different resolution levels in the brain region of interest (e.g., lobes, hemispheres). Patients' charateristics (i.e., occurrence and sparsity) might also be explained by the non-imaging data as well. Advantages of the current approach can be experienced by any patients (i.e., with or without imaging sessions) in any clinical facilities (i.e., with or without imaging instrumentation).

Silver-doped calcium silicate sol-gel glasses with a cotton-wool-like structure for wound healing.

Skin has excellent capacity to regenerate, however, in the event of a large injury or burn skin grafts are required to aid wound healing. The regenerative capacity further declines with increasing age and can be further exacerbated with bacterial infection leading to a chronic wound. Engineered skin substitutes can be used to provide a temporary template for the damaged tissue, to prevent/combat bacterial infection and promote healing. In this study, the sol-gel process and electrospinning were combined to fabricate 3D cotton-wool-like sol-gel bioactive glass fibers that mimic the fibrous architecture of skin extracellular matrix (ECM) and deliver metal ions for antibacterial (silver) and therapeutic (calcium and silica species) actions for successful healing of wounds. This study investigated the effects of synthesis and process parameters, in particular sintering temperature on the fiber morphology, the incorporation and distribution of silver and the degradation rate of fibers. Silver nitrate was found to decompose into silver nanoparticles within the glass fibers upon calcination. Furthermore, with increasing calcination temperature the nanoparticles increased in size from 3 nm at 600 °C to ~25 nm at 800 °C. The antibacterial ability of the Ag-doped glass fibers decreased as a function of the glass calcination temperature. The degradation products from the Ag-doped 3D non-woven sol-gel glass fibers were also found to promote fibroblast proliferation thus demonstrating their potential for use in skin regeneration.

Proposal of layered mental healthcare for mental well-being.

A new concept, 'Layered mental healthcare' for keeping employees mental well-being in the workplace to avoid losses caused by both absenteeism and presenteeism is proposed. A key factor forming the basis of the concept is the biometric measurements over three layers, i.e., behaviour, physiology, and brain layers, for monitoring mental/distress conditions of employees. Here, the necessity of measurements in three layers was validated by the data-driven approach using the preliminary dataset measured in the office environment. Biometric measurements were supported by an activity tracker, a PC logger, and the optical topography; mental/distress conditions were quantified by the brief job stress questionnaire. The biometric features obtained 1 week before the measurement of mental/distress scores were selected for the best regression model. The feature importance of each layer was obtained in the learning process of the best model using the light graded boosting machine and was compared between layers. The ratio of feature importance of behaviour:physiology:brain layers was found to be 4:3:3. The study results suggest the contribution and necessity of the three-layer features in predicting mental/distress scores.

Mental Condition Monitoring Based on Multimodality Biometry.

We have developed a system with multimodality that monitors objective biomarkers for screening the mental distress in the office. A field study using a prototype of the system was performed over four months with 39 volunteers. We obtained PC operation patterns using a PC logger, sleeping time and activity levels using a wrist-band-type activity tracker, and brain activity and behavior data during a working memory task using optical topography. We also administered two standard questionnaires: the Brief Job Stress Questionnaire (BJS) and the Kessler 6 scale (K6). Supervised machine learning and cross validation were performed. The objective variables were mental scores obtained from the questionnaires and the explanatory variables were the biomarkers obtained from the modalities. Multiple linear regression models for mental scores were comprehensively searched and the optimum models were selected from 2,619,785 candidates. Each mental score estimated with each optimum model was well correlated with each mental score obtained with the questionnaire (correlation coefficient = 0.6-0.8) within a 24% of estimation error. Mental scores obtained by means of questionnaires have been in general use in mental health care for a while, so our multimodality system is potentially useful for mental healthcare due to the quantitative agreement on the mental scores estimated with biomarkers and the mental scores obtained with questionnaires.

Lesions in the right Rolandic operculum are associated with self-rating affective and apathetic depressive symptoms for post-stroke patients.

Stroke survivors majorly suffered from post-stroke depression (PSD). The PSD diagnosis is commonly performed based on the clinical cut-off for psychometric inventories. However, we hypothesized that PSD involves spectrum symptoms (e.g., apathy, depression, anxiety, and stress domains) and severity levels. Therefore, instead of using the clinical cut-off, we suggested a data-driven analysis to interpret patient spectrum conditions. The patients' psychological conditions were categorized in an unsupervised manner using the k-means clustering method, and the relationships between psychological conditions and quantitative lesion degrees were evaluated. This study involved one hundred sixty-five patient data; all patients were able to understand and perform self-rating psychological conditions (i.e., no aphasia). Four severity levels-low, low-to-moderate, moderate-to-high, and high-were observed for each combination of two psychological domains. Patients with worse conditions showed the significantly greater lesion degree at the right Rolandic operculum (part of Brodmann area 43). The dissimilarities between stress and other domains were also suggested. Patients with high stress were specifically associated with lesions in the left thalamus. Impaired emotion processing and stress-affected functions have been frequently related to those lesion regions. Those lesions were also robust and localized, suggesting the possibility of an objective for predicting psychological conditions from brain lesions.

Electrospinning 3D bioactive glasses for wound healing.

An electrospinning technique was used to produce three-dimensional (3D) bioactive glass fibrous scaffolds, in the SiO2-CaO sol-gel system, for wound healing applications. Previously, it was thought that 3D cotton wool-like structures could only be produced from sol-gel when the sol contained calcium nitrate, implying that the Ca2+ and its electronic charge had a significant effect on the structure produced. Here, fibres with a 3D appearance were also electrospun from compositions containing only silica. A polymer binding agent was added to inorganic sol-gel solutions, enabling electrospinning prior to bioactive glass network formation and the polymer was removed by calcination. While the addition of Ca2+ contributes to the 3D morphology, here we show that other factors, such as relative humidity, play an important role in producing the 3D cotton-wool-like macrostructure of the fibres. A human dermal fibroblast cell line (CD-18CO) was exposed to dissolution products of the samples. Cell proliferation and metabolic activity tests were carried out and a VEGF ELISA showed a significant increase in VEGF production in cells exposed to the bioactive glass samples compared to control in DMEM. A novel SiO2-CaO nanofibrous scaffold was created that showed tailorable physical and dissolution properties, the control and composition of these release products are important for directing desirable wound healing interactions.

Tuning of ion-release capability from bio-ceramic-polymer composites for enhancing cellular activity.

In our previous study, we investigated the synergetic effects of inorganic ions, such as silicate, Mg2+ and Ca2+ ions on the osteoblast-like cell behaviour. Mg2+ ions play an important role in cell adhesion. In the present study, we designed a new composite that releases a high concentration of Mg2+ ions during the early stage of the bone-forming process, and silicate and Ca2+ ions continuously throughout this process. Here, 40SiO2-40MgO-20Na2O glass (G) with high solubility and vaterite-based calcium carbonate (V) were selected as the source of silicate and Mg2+ and Ca2+ ions, respectively. These particles were mixed with poly(lactic-co-glycolic acid) (PLGA) using a kneading method at 110°C to prepare the composite (G-V/PLGA, G/V/PLGA = 4/56/40 (in weight ratio)). Most of the Mg2+ ions were released within 3 days of immersion at an important stage for cell adhesion, and silicate and Ca2+ ions were released continuously at rates of 70-80 and 180 ppm d-1, respectively, throughout the experiment (until day 7). Mouse-derived osteoblast-like MC3T3-E1 proliferated more vigorously on G-V/PLGA in comparison with V-containing PLGA without G particles; it is possible to control the ion-release behaviour by incorporating a small amount of glass particles.

Prefrontal cortex activation of return-to-work trainees in remission of mental disorders with depressive symptoms compared to that of healthy controls.

The increase in the number of patients with mental disorders with depressive symptoms has become a significant problem. To prevent people developing those disorders and help with the effective recovery, it is important to quantitatively and objectively monitor an individual's mental state. Previous studies have shown the relationship between negative or depressive mood state and human prefrontal cortex (PFC) activation during verbal and spatial working memory tasks based on a near-infrared spectroscopy imaging technique. In this study, we aimed to explore a biomarker of the mental state of people in remission of mental disorders with depressive symptoms using this technique. We obtained the PFC activation of return-to-work (RTW) trainees in remission of those disorders, compared that of healthy controls, and obtained subjective questionnaire scores with the Profile of Mood States. We compared the PFC activation with the questionnaire scores by receiver operating characteristic analysis using a logistic-regression model. The results showed that the PFC activation indicates a healthy state compared to that of the RTW trainees evaluated by area-under-curve analysis. This study demonstrates that our PFC measurement technique will be useful as a quantitative and objective assessment of mental state.

Denoising of neuronal signal from mixed systemic low-frequency oscillation using peripheral measurement as noise regressor in near-infrared imaging.

Functional near-infrared spectroscopy (fNIRS) is a noninvasive functional imaging technique measuring hemodynamic changes including oxygenated ( O 2 Hb ) and deoxygenated (HHb) hemoglobin. Low frequency (LF; 0.01 to 0.15 Hz) band is commonly analyzed in fNIRS to represent neuronal activation. However, systemic physiological artifacts (i.e., nonneuronal) likely occur also in overlapping frequency bands. We measured peripheral photoplethysmogram (PPG) signal concurrently with fNIRS (at prefrontal region) to extract the low-frequency oscillations (LFOs) as systemic noise regressors. We investigated three main points in this study: (1) the relationship between prefrontal fNIRS and peripheral PPG signals; (2) the denoising potential using these peripheral LFOs, and (3) the innovative ways to avoid the false-positive result in fNIRS studies. We employed spatial working memory (WM) and control tasks (e.g., resting state) to illustrate these points. Our results showed: (1) correlation between signals from prefrontal fNIRS and peripheral PPG is region-dependent. The high correlation with peripheral ear signal (i.e., O 2 Hb ) occurred mainly in frontopolar regions in both spatial WM and control tasks. This may indicate the finding of task-dependent effect even in peripheral signals. We also found that the PPG recording at the ear has a high correlation with prefrontal fNIRS signal than the finger signals. (2) The systemic noise was reduced by 25% to 34% on average across regions, with a maximum of 39% to 58% in the highly correlated frontopolar region, by using these peripheral LFOs as noise regressors. (3) By performing the control tasks, we confirmed that the statistically significant activation was observed in the spatial WM task, not in the controls. This suggested that systemic (and any other) noises unlikely violated the major statistical inference. (4) Lastly, by denoising using the task-related signals, the significant activation of region-of-interest was still observed suggesting the manifest task-evoked response in the spatial WM task.

Combinatorial effects of inorganic ions on adhesion and proliferation of osteoblast-like cells.

Combinatorial effects of three ions, namely silicate (Si), calcium (Ca), and magnesium (Mg) ions, on the adhesion and proliferation of MC3T3-E1 mouse osteoblast-like cells were evaluated. The cells were cultured in single-, dual-, or triple-ion-conditioned culture media with systematically changed ion concentrations. The ranges of Si, Ca, and Mg ion concentrations were set as 10-70, 80-400, and 25-500 ppm, respectively. The numbers of adherent live cells were measured after culturing for 3 h and for 1, 3, and 5 days to examine cell adhesion and proliferation, respectively. Mg ions predominantly enhanced cell adhesion in both the dual-ion (xSi-zMg and yCa-zMg) and triple-ion (xSi-yCa-zMg) systems but had no effect when they acted individually in the single-ion system. Conversely, Si ions predominantly enhanced cell proliferation in most single- and triple-ion-conditioned media. Evaluation of the combinatorial effects of the three ions on cell adhesion and proliferation revealed that the dual- and triple-ion-conditioned media mainly conferred synergistic effects on adhesion but antagonistic effects on proliferation. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1042-1051, 2019.

Construction of DNAzyme-Encapsulated Fibermats Using the Precursor Network Polymer of Poly(γ-glutamate) and 4-Glycidyloxypropyltrimethoxysilane.

Here, we developed functional nucleic acid (FNA)-encapsulated electrospun fibermats. To facilitate stable FNA encapsulation in the γ-PGA/GPTMS fibermats, we used the FNA as an FNA/streptavidin complex, and as a representative FNA, we selected a DNAzyme, the DNA/hemin complex, which is composed of G-quadraplex-forming single-stranded DNA and hemin and exhibits oxidation activity with the aid of a cocatalyst, H2O2. Scanning electron microscopy and Fourier-transform infrared spectroscopy measurements revealed that encapsulation of the DNA/hemin complex (∼1 wt % against the γ-PGA/GPTMS hybrid) in the nanofibers of the γ-PGA/GPTMS fibermats did not affect the structure of the original nanofibers. However, because a unique MW-dependent molecular permeability originated from the 3D network structure of the γ-PGA/GPTMS hybrid, low-MW substrates such as 4-aminoantipyrine, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, and luminol were able to reach the encapsulated DNA/hemin complex by permeating to the inside of the nanofibers from an immersion buffer and then underwent catalytic oxidation. Conversely, nucleases, which are proteins featuring high MWs (>5 kDa), could not penetrate the γ-PGA/GPTMS nanofibers, and the encapsulated DNA/hemin complex was therefore effectively protected against nuclease digestion. Thus, encapsulating FNAs on the inside of the nanofibers of fibermats offers clear advantages for the practical application of FNAs in sensors and drugs, particularly for use in the in vivo circumstances.

Osteoblast-like cell responses to ion products released from magnesium- and silicate-containing calcium carbonates.

BACKGROUND: Inorganic ions released from bioceramics and bioactive glasses have been reported to influence osteogenic cell functions. Cell responses depend on types of the ions provided, for example, silicate ion has been found to up-regulate their proliferation, differentiation and mineralization. OBJECTIVE: Mouse osteoblast-like cells (MC3T3-E1) were cultured in media containing silicate and calcium ions with/without magnesium ion to evaluate their combined effects on the cell's functions. METHODS: The cells were cultured in the media containing the extract of silicate-containing vaterite (SiV) and magnesium- and siloxane-containing one (MgSiV) and normal medium and then their adhesion, proliferation, differentiation and mineralization were evaluated. RESULTS: The adhesion of the cells was enhanced when they were cultured in the medium containing MgSiV-extract. Their proliferation and differentiation were up-regulated in both media containing MgSiV-extract and SiV-extract. In particular, the MgSiV-extract significantly enhanced their differentiation than the SiV-extract. This was supported by the mineralization test's results, which showed a large amount of mineral deposit was observed in the cells cultured in the MgSiV-extract medium. CONCLUSIONS: Providing the three kinds of ions was effective for up-regulating the cell's mineralization compared to providing silicate and calcium ions without magnesium ion.

Tutorial on platform for optical topography analysis tools.

Optical topography/functional near-infrared spectroscopy (OT/fNIRS) is a functional imaging technique that noninvasively measures cerebral hemoglobin concentration changes caused by neural activities. The fNIRS method has been extensively implemented to understand the brain activity in many applications, such as neurodisorder diagnosis and treatment, cognitive psychology, and psychiatric status evaluation. To assist users in analyzing fNIRS data with various application purposes, we developed a software called platform for optical topography analysis tools (POTATo). We explain how to handle and analyze fNIRS data in the POTATo package and systematically describe domain preparation, temporal preprocessing, functional signal extraction, statistical analysis, and data/result visualization for a practical example of working memory tasks. This example is expected to give clear insight in analyzing data using POTATo. The results specifically show the activated dorsolateral prefrontal cortex is consistent with previous studies. This emphasizes analysis robustness, which is required for validating decent preprocessing and functional signal interpretation. POTATo also provides a self-developed plug-in feature allowing users to create their own functions and incorporate them with established POTATo functions. With this feature, we continuously encourage users to improve fNIRS analysis methods. We also address the complications and resolving opportunities in signal analysis.

Construction and Characterization of Protein-Encapsulated Electrospun Fibermats Prepared from a Silica/Poly(γ-glutamate) Hybrid.

Protein-encapsulated fibermats are an attractive platform for protein-based bioactive materials. However, the choice of methods is still limited and not applicable to a wide range of proteins. In this study, we studied new polymeric materials for constructing protein-encapsulated fibermats, in which protein molecules are encapsulated within the nanofibers of fibermats without causing deleterious changes to protein structure or function. We constructed a protein-encapsulated fibermat using the poly(γ-glutamate) (PGA)/(3-glycidyloxypropyl)-trimethoxysilane (GPTMS) hybrid as a precursor for electrospinning. Because the PGA/GPTMS hybrid is water-soluble, protein molecules can be added to the precursor in an aqueous solution, significantly enhancing protein stability. Polycondensation during electrospinning (in-flight polycondensation) makes the obtained fibermats water-insoluble, which stabilizes the fibermat structure such that it is resistant to degradation in aqueous buffer. The molecular structure of the PGA/GPTMS hybrid gives rise to unique molecular permeability, which alters the selectivity and specificity of biochemical reactions involving the encapsulated enzymes; lower molecular-weight (MW) substrates can permeate the nanofibers, promoting enzyme activity, but higher MW substrates such as inhibitor peptides cannot permeate the nanofibers, suppressing enzyme activity. We present an effective method of encapsulating bioactive molecules while maintaining their structure and function, increasing the versatility of electrospun fibermats for constructing various bioactive materials.

Development of Magnesium and Siloxane-Containing Vaterite and Its Composite Materials for Bone Regeneration.

Development of novel biomaterials with Mg(2+), Ca(2+), and silicate ions releasability for bone regeneration is now in progress. Several inorganic ions have been reported to stimulate bone-forming cells. We featured Ca(2+), silicate, and especially, Mg(2+) ions as growth factors for osteoblasts. Various biomaterials, such as ceramic powders and organic-inorganic composites, that release the ions, have been developed and investigated for their cytocompatibilities in our previous work. Through the investigation, providing the three ions was found to be effective to activate osteogenic cells. Magnesium and siloxane--containing vaterite was prepared by a carbonation process as an inorganic particle that can has the ability to simultaneously release Ca(2+), silicate, and Mg(2+) ions to biodegradable polymers. Poly (l-lactic acid) (PLLA)- and bioactive PLLA-based composites containing vaterite coatings were discussed regarding their degradability and cytocompatibility using a metallic Mg substrate as Mg(2+) ion source. PLLA/SiV composite film, which has a releasability of silicate ions besides Ca(2+) ion, was coated on a pure Mg substrate to be compared with the PLLA/V coating. The degradability and releasability of inorganic ions were morphologically and quantitatively monitored in a cell culture medium. The bonding strength between the coatings and Mg substrates was one of the key factors to control Mg(2+) ion release from the substrates. The cell culture tests were conducted using mouse osteoblast-like cells (MC3T3-E1 cells); cellular morphology, proliferation, and differentiation on the materials were evaluated. The PLLA/V and PLLA/SiV coatings on Mg substrates were found to enhance the proliferation, especially the PLLA/SiV coating possessed a higher ability to induce the osteogenic differentiation of the cells.

Impact of Cationic Amino Acid Transporter 1 on Blood-Retinal Barrier Transport of L-Ornithine.

PURPOSE: To elucidate L-ornithine transport at the blood-retinal barrier (BRB). METHODS: Integration plot and retinal uptake index (RUI) were used to investigate the in vivo [3H]L-ornithine transport across the BRB. In vitro transport studies of [3H]L-ornithine were performed with TR-iBRB2 cells and RPE-J cells, the model cells of the inner and outer BRB, respectively. Immunohistochemistry was performed on cationic amino acid transporter 1 (CAT1/SLC7A1). RESULTS: The apparent influx permeability clearance of [3H]L-ornithine was found to be 18. 7 µL/(min·g retina), and the RUI of [3H]L-ornithine was reduced by L-ornithine and L-arginine, suggesting the blood-to-retina transport of L-ornithine at the BRB. [3H]L-Ornithine uptake by TR-iBRB2 cells showed a time-, temperature- and concentration-dependence with a Michaelis-Menten constant (K(m)) of 33.2 µM and a nonsaturable uptake rate (Kd) of 2.18 µL/(min·mg protein). The uptake was Na+-independent, and was inhibited by L-ornithine, L-arginine, and L-lysine, suggesting the involvement of CAT1 in L-ornithine transport at the inner BRB. Immunohistochemistry revealed the luminal and abluminal localization of CAT1 at the inner BRB, and at the basal localization at the outer BRB. Retinal pigment epithelium-J cells showed that the basal-to-cell (B-to-C) uptake of [3H]L-ornithine was greater than that of the apical-to-cell (A-to-C) uptake, and the B-to-C transport was inhibited by unlabeled L-ornithine, suggesting the involvement of CAT1 in the blood-to-cell transport of L-ornithine across the basal membrane at the outer BRB. CONCLUSIONS: These suggest the involvement of CAT1 in L-ornithine transport at the luminal and abluminal sides of the inner BRB and the basal side of the outer BRB.

Preparation of Cotton-Wool-Like Poly(lactic acid)-Based Composites Consisting of Core-Shell-Type Fibers.

In previous works, we reported the fabrication of cotton-wool-like composites consisting of siloxane-doped vaterite and poly(l-lactic acid) (SiVPCs). Various irregularly shaped bone voids can be filled with the composite, which effectively supplies calcium and silicate ions, enhancing the bone formation by stimulating the cells. The composites, however, were brittle and showed an initial burst release of ions. In the present work, to improve the mechanical flexibility and ion release, the composite fiber was coated with a soft, thin layer consisting of poly(d,l-lactic-co-glycolic acid) (PLGA). A coaxial electrospinning technique was used to prepare a cotton-wool-like material comprising "core-shell"-type fibers with a diameter of ~12 µm. The fibers, which consisted of SiVPC coated with a ~2-µm-thick PLGA layer, were mechanically flexible; even under a uniaxial compressive load of 1.5 kPa, the cotton-wool-like material did not exhibit fracture of the fibers and, after removing the load, showed a ~60% recovery. In Tris buffer solution, the initial burst release of calcium and silicate ions from the "core-shell"-type fibers was effectively controlled, and the ions were slowly released after one day. Thus, the mechanical flexibility and ion-release behavior of the composites were drastically improved by the thin PLGA coating.