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Background: Epidural catheters are associated with certain risks such as accidental epidural catheter removal, including dislodgement and disconnection. Globally, neuraxial connector designs were revised in 2016 to provide new standardization aimed at decreasing the frequency of misconnections during the administration of medications. However, no studies have investigated accidental epidural catheter removal after the revised standardization. This study aimed to examine differences in dislodgement and disconnection rates associated with different catheter connector types, and to investigate the linear tensile strength required to induce disconnection., Methods: This retrospective cohort study included adult patients who underwent elective surgery and received patient-controlled epidural analgesia. Patients were divided into groups according to the type of catheter connection used: old standard, new standard, and new standard with taping groups. Furthermore, we prepared 60 sets of epidural catheters and connectors comprising 20 sets for each of the old, new, and taping groups, and used a digital tension meter to measure the maximum tensile strength required to induce disconnection., Results: This clinical study involved 360, 182, and 378 patients in the old, new, and taping groups, respectively. Dislodgement rates did not differ statistically among the three groups, while there was a significant difference in disconnection rates. Propensity score matching analysis for disconnection rates showed no difference between the old and new groups (2.8% vs. 4.5%, p = 0.574), while the new group had higher rates than the taping group (6.5% vs. 0%, p = 0.002). This laboratory study identified that a tensile strength of 12.41 N, 12.06 N, and 19.65 N was required for disconnection in the old, new, and taping groups, respectively, and revealed no significant difference between the new and old groups (p = 0.823), but indicated a significant difference between the new and taping groups (p < 0.001)., Conclusions: This clinical study suggested that dislodgement rates did not change among the three groups. Both clinical and laboratory studies revealed that disconnection rates did not change between the old and new connectors. Moreover, as a strategy to prevent accidents, taping the connecting points of the catheter connectors led to an increase in the tensile strength required for disconnection., (© 2022. The Author(s).)

Background: Maternal diet and sociodemographic factors influence xanthophyll concentration and composition in human milk. However, the importance of dietary patterns regarding the intake of fruits, vegetables, and xanthophylls remains unclear., Objective: The aim was to determine the composition of xanthophylls in the human milk of Japanese mothers and explore associations of xanthophylls with dietary and sociodemographic factors., Methods: This cross-sectional study was conducted in the early phase of the Japanese Human Milk Study. Xanthophyll content was measured using liquid chromatography at 30-36 d postpartum. Maternal intake of foods, nutrients, and dietary supplements was estimated using a food-frequency questionnaire. Linear regression models were established using xanthophylls, maternal diet, and sociodemographic factors., Results: Xanthophyll concentrations were measured in human milk from 118 mothers. The xanthophyll concentration varied among individuals. The median (IQR) concentrations of lutein, zeaxanthin, and β-cryptoxanthin were 65.6 ng/mL (51.6-103.4 ng/mL), 18.6 ng/mL (12.9-25.8 ng/mL), and 15.6 ng/mL (9.0-26.0 ng/mL), respectively. In multivariate models, the lutein concentration was associated independently with dietary green vegetables, exclusive breastfeeding, and education ( r 2 = 0.153 for the model; β ± SE: 0.468 ± 0.198, 25.048 ± 10.222, and 13.460 ± 6.774; standardized β = 0.210, 0.217, and 0.175; P = 0.019, 0.016, and 0.049 for dietary green vegetables, exclusive breastfeeding, and education, respectively). For zeaxanthin, exclusive breastfeeding was the most appropriate predictor ( r 2 = 0.085; β ± SE: 7.811 ± 3.300; standardized β = 0.218; P = 0.020). The highest predictive power for human milk β-cryptoxanthin was obtained with dietary β-cryptoxanthin ( r 2 = 0.258; β ± SE: 0.089 ± 0.015; standardized β = 0.468; P < 0.001), attributed to maternal citrus intake., Conclusions: β-Cryptoxanthin in human milk was the xanthophyll most influenced by the maternal diet in Japanese women. The β-cryptoxanthin concentration in human milk was reflected by the maternal β-cryptoxanthin intake, mainly attributed to Japanese citrus consumption. This trial was registered in the Japanese Clinical Trials Registry (https://center6.umin.ac.jp/cgi-open-bin/ctr&#95;e/ctr&#95;view.cgi?recptno=R000017649) as UMIN000015494., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society for Nutrition.)

This study investigated whether parenting stress is associated with breastfeeding type (exclusive or partial). Between 2014 and 2019, we recruited 1210 healthy mothers (mean age, 31.2 years; 65%, multiparity) from 73 obstetric institutions across all prefectures of Japan. Among these, 1120 mothers at two months and 1035 mothers at six months were investigated for parenting stress and breastfeeding type: exclusive versus otherwise (partial). Parenting stress was measured by a validated Japanese scale consisting of childcare exhaustion, worry about child's development, and no partner support. Exclusive breastfeeding prevalence was 75% at two and 78% at six months. The total scores for childcare exhaustion and worry about child development were statistically higher in the partial breastfeeding group than in the exclusive breastfeeding group at two months but not at six months. A logistic regression model demonstrated that childcare exhaustion was significantly associated with an increased risk of having partial breastfeeding at two months after adjusting for the maternal Body Mass Index, parity, and baby's current weight. However, the association was no longer significant at six months. The present study suggests that intervention for parenting stress at two months postpartum may promote prolonged exclusive breastfeeding.

Background: Despite a number of studies comparing laparoscopic inguinal hernia repair (LH) and open herniorrhaphy (OH), the putative advantage of LH remains controversial due to a paucity of firm evidence. We hypothesized that LH has both advantages and disadvantages compared to OH and sought to clarify them by comprehensively analyzing the retrospective data using the combination of multiple statistical methods., Methods: Operative data for inguinal hernia during the period from February 1999 to December 2019 were examined. The patients were assigned into two groups according to the surgical procedure: laparoscopic percutaneous extraperitoneal closure (LPEC, n = 2410) and OH (n = 2038). Operative and anesthesia times and incidence of postoperative complications were evaluated using the propensity score methods and log-rank test., Results: In comparison with OH, operative time of LPEC was longer for unilateral repair (21.59 ± 8.1 min vs 18.01 ± 8.0 min; p < 0.001) and shorter for bilateral repairs (28.55 ± 10.1 min vs 33.23 ± 11.7 min; p < 0.001), while anesthesia times were longer for both unilateral repair (57.67 ± 10.1 min vs 40.62 ± 11.9 min; p < 0.001) and bilateral repairs (65.95 ± 12.5 min vs 56.35 ± 15.1 min; p < 0.001). LPEC significantly reduced the risk of metachronous contralateral hernia (MCLH) (0.52% vs 9.29%; p < 0.001), but the recurrence rate was higher (0.21% vs 0.04%; p = 0.002) than OH. Orchiectomy due to testicular atrophy or torsion was required in 3 cases of OH (0.19%), whereas it was not seen in LPEC., Conclusions: LPEC had a less risk of MCLH and testicular complications but was associated with a higher recurrence rate and longer anesthesia time. Propensity scoring techniques can enhance the robustness of retrospective comparisons between groups over several years of data collection, which is frequently required in pediatric surgery studies., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

Purpose: The Japanese Human Milk Study, a longitudinal prospective cohort study, was set up to clarify how maternal health, nutritional status, lifestyle and sociodemographic and economic factors affect breastfeeding practices and human milk composition. This would eventually determine factors affecting the growth and development of infants and children., Participants: A total of 1210 Japanese lactating women who satisfied the inclusion criteria, were invited across the country at various participating sites, between 2014 and 2019. Finally a total of 1122 women were enrolled in this study., Findings to Date: Among 1122 eligible participants, mean age at delivery was 31.2 (SD 4.4) years and mean prepregnancy BMI was 20.8 (SD 2.7). Among these women, 35% were previously nulliparous and 77.7% had college, university or higher education. The mean gestational period was 39.0 (SD 1.3) weeks. Caesarean section was reported among 11.9%; mean infant birth weight was 3082 (SD 360) g. Of the infants, 53.7% were male. Overall, our participants appeared to be healthier than the general population in Japan. Analyses of the 1079 eligible human milk samples obtained at the first and second months postpartum showed the following composition: carbohydrate, 8.13 (SD 0.32) g/100 mL; fat, 3.77 (SD 1.29) g/100 mL; and crude protein, 1.20 (SD 0.23) g/100 mL. We also analysed osteopontin, fatty acid, vitamin D and phospholipid levels in limited subcohorts of the samples., Future Plans: Follow-up surveys will be conducted to obtain milk samples every 2 months for 12 months and to investigate mother and child health until the children reach 5 years of age. These will be completed in 2024. We plan to longitudinally analyse the composition of macronutrients and various bioactive factors in human milk and investigate the lifestyle and environmental factors that influence breastfeeding practices, maternal and child health, and child development., Trial Registration Number: UMIN000015494; pre-results., Competing Interests: Competing interests: KeN, SH, TT, YTs and YTo are employees of Bean Stalk Snow. SK, J-iY and HMU were past employees of Bean Stalk Snow and currently employees of Megmilk Snow Brand. Bean Stalk Snow manufactured and sold infant formula and dietary supplements for pregnant and lactating women in Japan. Megmilk Snow Brand manufactured and sold infant formula in Japan, Taiwan, Hong Kong and Malaysia. The other author declares no conflicts of interest., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Various three-dimensional (3D) culture systems are available to provide more accurate in vivo mimicry than two-dimensional (2D) cultures. Synthetic and/or xeno-free biomaterials are desired, as they would provide lower batch-to-batch variability and high repeatability. Here, we introduce a 3D culture system using nanofibers composed of an amphiphilic polydepsipeptide-based polymer named HYDROX, which turns into 3D nanofibers after hydration. Our system produces a large amount of cell aggregates and requires only the seeding of a cell mixture. In addition, cells cultured with HYDROX can be collected with only a centrifugation procedure, and analytical assays can then be performed. Here, we applied HYDROX to hepatic differentiation from induced pluripotent stem cells. The cells cultured with HYDROX formed aggregates and HYDROX strongly promoted hepatic differentiation and maturation in terms of functions such as the positive ratio of alpha-1 antitrypsin, the production of albumin, the cytochrome P450 (CYP) 3A4 activity, and the low-density-lipoprotein uptake ability. In addition, primary human hepatocytes cultured with HYDROX showed significantly improved CYP3A4 gene expression and activity. The viscoelasticity and stiffness of HYDROX can be modulated by varying the concentration of the synthetic polymer, thereby providing a suitable microenvironment for the differentiation of cells with various characteristics toward a target cell type. Our findings demonstrated that HYDROX is a promising biomaterial for 3D cultures in research fields ranging from basic cell research to drug discovery.

Hip fracture is a common injury in elderly patients. In Japan, the number of super-old patients-age >90 years-with hip fractures has increased drastically over time. Available strategies for anaesthetic management for hip fracture surgery include general anaesthesia, neuraxial anaesthesia and peripheral nerve block. However, general and neuraxial anaesthesia are often avoided for various reasons, particularly in elderly patients. In recent years, peripheral nerve block has proven effective in various surgical procedures. Additionally, dexmedetomidine exhibits neuroprotective effects and has been used safely in super-old patients. Herein, we demonstrate successful anaesthetic management with peripheral nerve block under dexmedetomidine sedation for open reduction and internal fixation of a femoral neck fracture in a 97-year-old patient., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

To characterize the milk phospholipids (PLs) profile using phosphorus-31 nuclear magnetic resonance ( 31 P-NMR) and to investigate the effect of elevated milk docosahexaenoic acid (DHA) levels on PLs profiles in Japanese mothers., Methods: Milk samples from eligible patients with high and low DHA from a former cross-sectional study (n = 20; n = 10 for each group) were included. Fifteen milk PLs were analyzed using 31 P-NMR, and the profiles were compared group-wise using Mann-Whitney U -test. The P value of <0.05 was considered statistically significant., Results: The median DHA content in milk was 1.13% and 0.29% for the high and low milk DHA groups, respectively. Twelve PLs, excluding lysophosphatidylserine, cardiolipin, and phosphatidylglycerol, were detected in all participants with 100% positive results. The median concentrations and proportions of total PLs, sphingophospholipids, and glycerophospholipids were comparable between groups. The proportions of choline-containing glycerophospholipid were significantly higher in the high milk DHA group than that in the low milk DHA group (24.09% [median, interquartile range: 23.08%-26.38%] and 21.41% [20.74%-22.84%], P = 0.019). Although the proportions of phosphatidylinositol were significantly lower in the high milk DHA group than that in the low milk DHA group (6.62% [5.75%-6.72%] versus 7.63% [7.11%-8.16%], P = 0.002), while that of phosphatidylcholine (21.90% [18.51%-23.22%] versus 19.78% [18.17%-20.26%], P = 0.059) and alkyl-acyl phosphatidylcholine (0.60% [0.40%-0.74%] versus 0.33% [0.14%-0.51%], P = 0.059) were higher in the former than that in the latter., Conclusions: Our results were comparable to that of the previous literature. Large variations in the milk DHA might affect the composition of choline-containing glycerophospholipids in Japanese mothers. However, possible confounders were not excluded in the study populations., Competing Interests: Drs Ueno, Higurashi, Toba, and Kobayashi are employees of Bean Stalk Snow. Bean Stalk Snow manufactures and sells infant formula and dietary supplements for pregnant and lactating women in Japan. The remaining authors report no conflicts of interest., (Copyright © 2021 The Author(s). Published by Wolters Kluwer on behalf of European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition.)

Objective: We investigated the relationship between gross motor abilities and sensory processing in typically developing children., Method: Participants included children aged 18 to 36 months (N = 48). All participants were full-term infants. We assessed gross motor abilities based on the Gross Motor Function Measure (GMFM), and sensory processing characteristics based on the Infant/Toddler Sensory Profile (ITSP). The gross motor ability index was calculated using GMFM score which was estimated from the age. Pearson's product moment correlation coefficients were used to examine the relationships between the gross motor ability indexes and ITSP section scores., Results: Our findings showed that gross motor ability may be related to oral sensory processing. The children who were more responsive to oral sensory processing tended to exhibit gross motor abilities below the standard for that age., Conclusion: Gross motor abilities were related with sensory processing, especially oral sensory processing, in children aged 18 to 36 months., Competing Interests: The authors disclose no conflicts of interest., (2021, JAPANESE PHYSICAL THERAPY ASSOCIATION.)

This study assessed the color improvement via zinc protoporphyrin IX (ZnPP) formation in nitrite-free, dry-cured sausages processed using five varieties of ZnPP-forming lactic acid bacteria (LAB). The ZnPP contents and color intensity of the sausages and other technological properties were analyzed during the processing of sausages. LAB count and acidity significantly increased in the LAB-inoculated sausages compared to the control group. The bright red color was observed both inside and outside the sausages inoculated with Lactococcus lactis subsp. cremoris and Leuconostoc lactis . However, a brown color was observed on the surface of the sausage inoculated with Lactobacillus spp. The redness of Lactococcus lactis subsp. cremoris -inoculated sausages was close to that of the nitrite-added group. Moreover, the external bright red color was improved by Lactococcus lactis subsp. cremoris due to the aerobic formation of ZnPP. Therefore, Lactococcus lactis subsp. cremoris can be used to improve the color of fermented meat products.

Introduction: In single-space combined spinal-epidural anesthesia (CSEA), it is important to correctly determine if the fluid coming out of the spinal needle is cerebrospinal fluid (CSF) or the liquid used in the loss of resistance (LOR) technique. In this study, we used mepivacaine for LOR and measured the pH values of CSF and mepivacaine to determine whether the pH test is a reliable method to confirm CSF when performing single-space CSEA., Methods: This clinical trial included 47 full-term pregnant women who underwent cesarean section. Single-space CSEA was administered at the lumbar intervertebral space using a small amount of mepivacaine for LOR. The pH values of CSF and mepivacaine were determined by the color of the test strip immediately after dropping. The area under the curve (AUC) for the pH values was calculated to determine the cutoff value for distinguishing between CSF and mepivacaine., Results: The median pH values were 7.7 (7.1-8.0) and 6.2 (5.9-6.8) for CSF and mepivacaine, respectively. When the cutoff value of pH for distinguishing CSF from mepivacaine was 7.1 or greater, the AUC was 1.0 (100% sensitivity and specificity). Our result demonstrated that CSF can be correctly distinguished from mepivacaine in patients undergoing cesarean section under single-space CSEA using a cutoff value of pH 7.1., Conclusion: The pH test is a simple and reliable method to confirm CSF when performing single-space CSEA with mepivacaine for LOR., Trial Registration: Accuracy of pH test paper for cerebrospinal fluid during spinal anesthesia: prospective study in healthy pregnant women under scheduled caesarean section; University Hospital Medical Information Network, UMIN000036454 . Registered 1 May 2019.

Background and Purpose: Paroxysmal atrial fibrillation (PAF) has been suggested as a major cause of embolic stroke of undetermined source (ESUS). Transient atrial mechanical dysfunction (stunning) frequently occurs after conversion of atrial fibrillation to sinus rhythm. The study aim was to determine if reversible atrial mechanical dysfunction in ESUS could help elucidate the mechanism of stroke., Methods: Eighty-five consecutive patients with acute ischemic stroke were enrolled according to the following inclusion criteria: [1] ≥55 years old; [2] normal sinus rhythm upon admission; [3] no apparent embolic source; and [4] transthoracic echocardiographic evaluation had been performed in both the early phase (<72 h) and late phase (>7 days) after stroke onset. There were 27 patients in the lacunar or atherothrombotic infarction group (controls), 22 in the PAF group, and 36 in the ESUS group. To determine atrial stunning, transmitral flow velocity profiles (Doppler peak E- [early diastolic] and A- [atrial systolic] waves) were obtained., Results: In the early phase, an E/A velocity ratio ≥ 1.0 was less common in the control group (1 patient, 3.7%) than in the PAF group (19 patients, 86.4%; p < 0.001) and ESUS group (10 patients, 27.8%; p < 0.05). In the late phase, the E/A ratio decreased to less than 1.0 in six patients (31.6%) who had PAF and in eight patients (80.0%) who had ESUS., Conclusion: Transient atrial mechanical dysfunction could be a helpful finding for elucidating the stroke mechanism in patients with ESUS, and early echocardiographic assessment could improve its detection., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest associated with this manuscript., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: DHA (22:6n-3) is essential for neurodevelopment in children, and its concentration in human breast milk is historically high in Japan. Dietary patterns in Japan might affect the fatty acid (FA) composition among lactating mothers., Objectives: This study aimed to characterize the composition of milk FAs and to identify any dietary and sociodemographic factors associated with the variability of DHA concentration in breast milk in the Japanese population., Methods: This cross-sectional study was performed as part of the Japanese Human Milk Study. Milk FAs were analyzed by GC at 1-6 mo postpartum, and maternal diet was estimated using an FFQ, including 11 types and cooking methods of seafoods, and the use of DHA supplements. The association of milk DHA with maternal diet and sociodemographic factors was investigated., Results: Milk FA concentrations were measured in 78 mothers, including 24 who routinely used DHA supplements. The DHA concentration in milk (overall median: 0.62%; IQR: 0.47%-0.78%) was higher in women who took DHA supplements than in women who had never used DHA supplements (0.74%compared with 0.55%; P  = 0.011). A linear regression model showed the association of milk DHA concentration with maternal dietary intake of grilled fish (β ± SE: 0.006 ± 0.003; standardized β: 0.234; r 2  = 0.232, P  = 0.036) after adjustment for DHA supplementation status, maternal and infant age, maternal BMI, and infant birth weight. Other FA concentrations were consistent, whereas caproic acid (6:0), undecylic acid (11:0), pentadecylic acid (15:0), palmitoleic acid (16:1n-7), and vaccenic acid (18:1n-7) varied by DHA supplementation status., Conclusions: The DHA concentration in human milk may be influenced by maternal grilled fish consumption and frequent DHA supplementation in lactating Japanese women. Milk DHA concentrations may reflect a dietary habit in Japanese mothers.This trial was registered at www.umin.ac.jp/ctr as UMIN000015494., (Copyright © The Author(s) on behalf of the American Society for Nutrition 2020.)

It is unknown whether maternal health and nutrition are related to human milk composition or growth and development of infants and children. Here, we describe a protocol for a prospective five-year cohort study to clarify (i) how maternal health and nutrition, socioeconomic factors, and lifestyles affect human milk composition, and (ii) whether these are associated with growth and development of infants and children. In our study, we recruited 1210 Japanese mothers with singleton pregnancies from 73 obstetrics clinics and hospitals across Japan, between 2014 and 2019. We will measure the following: health information regarding maternal-child dyads using a self-administered questionnaire, maternal nutrition during breastfeeding using a Brief self-administrated Diet History Questionnaire, the development of infants and children using the Kinder Infant Development Scale, and the stress related to child rearing using the Mother's Child Care Stress Scale. Simultaneously, we will collect human milk every 2 months during the first year after birth to measure its composition and levels of macronutrients. This study will generate useful data to investigate whether health status, nutritional status, lifestyle, and socioeconomic factors affect human milk composition and the growth and development of infants and children.

Human hepatocytes are essential materials in pharmaceutical researches. Not only primary human hepatocytes (PHH) but also human iPS cell-derived hepatocyte-like cells (human iPS-HLCs) are expected to be applied as materials for pharmaceutical researches. To date, several culture media have been developed for culturing human hepatocytes. However, there have been no reports comparing these media to determine which is most suitable for culturing human hepatocytes. In this study, we compared five commercial media (Hepatocyte Culture Medium (HCM), HepatoZYME-SFM, Cellartis Power Primary HEP Medium, DMEM/F12, and William's E Medium (WEM)) to determine which is most suitable for culturing PHH and human iPS-HLCs. In hepatic differentiation of human iPS cells (day 14-25 of differentiation), albumin (ALB) and urea secretion abilities and CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM or WEM. During maintenance of human iPS-HLCs, ALB and urea producing abilities and CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM. Importantly, we found that human iPS-HLCs cultured in HCM were maintained for 3 weeks or more without impairment of their hepatic functions. These results suggest that it is necessary to select an optimal medium for hepatic differentiation and maintenance of human iPS-HLCs. In the case of PHH culture, there was little difference in hepatic functions among the five media. However, the CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM and WEM. In conclusion, it is important to select the optimal medium for specific application when carrying out pharmaceutical researches using human hepatocytes., Competing Interests: The authors have declared that no competing interests exist.

Single nucleotide polymorphisms in Tolloid-like 1 (TLL1) and the expression of TLL1 are known to be closely related to hepatocarcinogenesis after hepatitis C virus elimination or liver fibrosis in patients with nonalcoholic fatty liver disease. TLL1 is a type of matrix metalloprotease and has two isoforms in humans, with the short isoform showing higher activity. However, the functional role of TLL1 in human liver development is unknown. Here, we attempted to elucidate the function of human TLL1 using hepatocyte-like cells generated from human pluripotent stem cells. First, we generated TLL1-knockout human induced pluripotent stem (iPS) cells and found that hepatic differentiation was promoted by TLL1 knockout. Next, we explored TLL1-secreting cells using a model of liver development and identified that kinase insert domain receptor (FLK1)-positive cells (mesodermal cells) highly express TLL1. Finally, to elucidate the mechanism by which TLL1 knockout promotes hepatic differentiation, the expression profiles of transforming growth factor beta ( TGFβ ), a main target gene of TLL1, and its related genes were analyzed in hepatic differentiation. Both the amount of active TGFβ and the expression of TGFβ target genes were decreased by TLL1 knockout. It is known that TGFβ negatively regulates hepatic differentiation. Conclusion: TLL1 appears to negatively regulate hepatic differentiation of human iPS cells by up-regulating TGFβ signaling. Our findings will provide new insight into the function of TLL1 in human liver development., (© 2020 The Authors. Hepatology Communications published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.)

Cytochrome P450 family 2 subfamily C member 19 (CYP2C19), in liver, plays important roles in terms of drug metabolism. It is known that CYP2C19 poor metabolizers (PMs) lack CYP2C19 metabolic capacity. Thus, unexpected drug-induced liver injury or decrease of drug efficacy would be caused in CYP2C19 substrate-treated CYP2C19 PMs. However, it is difficult to evaluate the safety and effectiveness of drugs and candidate compounds for CYP2C19 PMs because there is currently no model for this phenotype. Here, using human induced pluripotent stem cells (human iPS cells) and our highly efficient genome-editing and hepatocyte differentiation technologies, we generated CYP2C19-knockout human iPS cell-derived hepatocyte-like cells (CYP2C19-KO HLCs) as a novel CYP2C19 PM model for drug development and research. The gene expression levels of hepatocyte markers were similar between wild-type iPS cell-derived hepatocyte-like cells (WT HLCs) and CYP2C19-KO HLCs, suggesting that CYP2C19 deficiency did not affect the hepatic differentiation potency. We also examined CYP2C19 metabolic activity by measuring S -mephenytoin metabolites using ultra-performance liquid chromatography-tandem mass spectrometry. The CYP2C19 metabolic activity was almost eliminated by CYP2C19 knockout. Additionally, we evaluated whether clopidogrel (CYP2C19 substrate)-induced liver toxicity could be predicted using our model. Unexpectedly, there was no significant difference in cell viability between clopidogrel-treated WT HLCs and CYP2C19-KO HLCs. However, the cell viability in clopidogrel- and ketoconazole (CYP3A4 inhibitor)-treated CYP2C19-KO HLCs was significantly enhanced as compared with that in clopidogrel- and DMSO-treated CYP2C19-KO HLCs. This result suggests that CYP2C19-KO HLCs can predict clopidogrel-induced liver toxicity. We succeeded in generating CYP2C19 PM model cells using human iPS cells and genome-editing technologies for pharmaceutical research. SIGNIFICANCE STATEMENT: Although unexpected drug-induced liver injury or decrease of drug efficacy would be caused in CYP2C19 substrate-treated CYP2C19 poor metabolizers, it is difficult to evaluate the safety and effectiveness of drugs and candidate compounds for CYP2C19 poor metabolizers because there is currently no model for this phenotype. Using human iPS cells and our highly efficient genome editing and hepatocyte differentiation technologies, we generated CYP2C19-knockout human iPS cell-derived hepatocyte-like cells as a novel CYP2C19 poor metabolizer model for drug development and research., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Human induced pluripotent stem cell-derived hepatocyte-like cells are expected to be utilized in pharmaceutical research and regenerative medicine. In general, human induced pluripotent stem (iPS) cells are differentiated into hepatocyte-like cells through definitive endoderm cells and hepatoblast-like cells using various growth factors that are essential for liver development. Although recombinant bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs) are widely used in the hepatoblast differentiation, hepatoblast differentiation process has not been fully modified. In this study, we examined the roles of BMPs and FGFs in the hepatoblast differentiation from human iPS cells. Surprisingly, the gene expression levels of hepatoblast markers were upregulated by the removal of FGFs. In addition, the percentages of hepatoblast markers-positive cells were increased by the removal of FGFs. Furthermore, the hepatocyte differentiation potency was also significantly increased by the removal of FGFs. To examine whether FGF signals are completely unnecessary for the hepatoblast differentiation, the expression levels of endogenous FGF ligands and receptors were examined. The definitive endoderm cells highly expressed the FGF ligand, FGF2, and the FGF receptor, FGFR1. To examine the role of endogenous FGF signals, an FGFR inhibitor was treated during the hepatoblast differentiation. The hepatoblast differentiation was promoted by using FGFR inhibitor, suggesting that endogenous FGF signals are also unnecessary for the hepatoblast differentiation. In conclusion, we found that FGF signals are not essential for hepatoblast differentiation. We believe that our finding will be useful for generating functional hepatocyte-like cells for medical applications.

Background: Autologous epidural blood patch (AEBP) is effective for post-dural-puncture headache (PDPH). In some cases, repeat procedures are required for complete cure. In rare instances, severe adverse effects can occur. We present a case of neurologically complicated AEBPs, one of which was performed at the interspace of unintentional dural puncture (UDP)., Case Presentation: A 40-year-old primigravida sustained UDP at the L2-3 interspace during combined spinal-epidural anesthesia for a scheduled cesarean section. She developed PDPH and underwent a single AEBP at L3-4. The PDPH recurred and she required another AEBP at L2-3, after which she reported radicular pains. A diagnosis of subdural hematoma and adhesive arachnoiditis was made. Her symptoms partially resolved in the following months., Conclusion: It may be prudent to reconsider the use of repeated AEBP and to avoid the interspace of UDP. A thorough evaluation is warranted to exclude treatable lesions when adverse effects occur.

Human iPS cell-derived hepatocyte-like cells are expected to be utilized in pharmaceutical research. However, the purity of high-functioning hepatocyte-like cells is not high enough. In particular, the purity of cytochrome P450 3A4 (CYP3A4), which is a representative hepatic drug-metabolizing enzyme, positive cells is still quite low (approximately 20%). To address this problem, we established the CYP3A4-NeoR-EGFP transgenic reporter human iPS cell line (CYP3A4-NeoR-EGFP iPS cells) by using genome editing technology. The CYP3A4-NeoR-EGFP iPS cells were differentiated into hepatocyte-like cells, and then the hepatocyte-like cells were treated with neomycin to concentrate the hepatocyte-like cells which strongly express CYP3A4. After the neomycin treatment, the percentage of CYP3A4-positive cells was higher than 80%. The gene expression levels of various drug-metabolizing enzymes, transporters, and hepatic transcription factors were significantly enhanced by neomycin treatment. In addition, the CYP1A2, 2C19, 2D6, and 3A4 activities and biliary excretion capacities were significantly increased by neomycin treatment. We also confirmed that the detection sensitivity of drug-inducing hepatotoxicity was enhanced by neomycin treatment. We succeeded in obtaining human iPS cell-derived hepatocyte-like cells that highly express CYP3A4 at high purity. We believe that our high-purity and high-functioning hepatocyte-like cells could be used to evaluate the risk of drug candidates., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Human induced pluripotent stem cell-derived hepatocyte-like cells (HLCs) are expected to be utilized in pharmaceutical research, including drug screening. However, the hepatocyte functions of the HLCs are still lower than those of human hepatocytes. Therefore, we attempted to improve the hepatocyte differentiation method by modulating the DNA epigenetic status. We first examined the expression profiles of the maintenance DNA methyltransferase (DNMT) 1 and the de novo DNMTs DNMT3A and DNMT3B, all of which are essential for mammalian development. Among these DNMTs, the expression levels of DNMT3B were significantly decreased during the hepatoblast differentiation. To accelerate the hepatoblast differentiation, a DNMT3B-selective inhibitor, nanaomycin A, was treated during the hepatoblast differentiation. The gene expression levels of hepatoblast markers (such as alpha-fetoprotein and hepatocyte nuclear factor 4 alpha) were increased by the nanaomycin A treatment. On the other hand, the gene expression levels of hepatoblast markers were decreased by DNMT3B overexpression. These results suggest that it might be possible to promote the hepatoblast differentiation by DNMT3B inhibition using nanaomycin A. Importantly, we also confirmed that the hepatocyte differentiation potency of nanaomycin A-treated hepatoblast-like cells was higher than that of dimethyl sulfoxide-treated hepatoblast-like cells. Our findings should assist in the future generation of functional HLCs for pharmaceutical research.

Middle cerebral artery (MCA) aneurysms usually arise at the primary MCA bifurcation or trifurcation. Distal MCA aneurysms are rarely considered as sources of aneurysmal subarachnoid hemorrhage (SAH). It has been reported that ruptured distal MCA aneurysms are associated with head trauma, neoplastic emboli, arterial dissection, or bacterial infection. We experienced five cases of ruptured distal MCA aneurysms and evaluated their clinical characteristics. Retrospective analysis of aneurysmal SAH at Kobayashi Neurosurgical Neurological Hospital was performed from January, 2004 to December, 2014. Clinical characteristics of ruptured distal MCA aneurysms were analyzed using our database. Among 191 aneurysmal SAH patients, there were five ruptured distal MCA aneurysms. All patients did not have any specific medical problems such as infectious disease, head trauma, or cardiac disorders. The incidence of ruptured distal MCA aneurysm was higher than expected and was equivalent to 9.4% of the total ruptured MCA aneurysms. Strong male predominance (80%) and M2-3 junction aneurysm preponderance (80%) were observed. In addition, there were only two patients (40%) with intracerebral hematoma in our study. We reported five cases of ruptured distal MCA aneurysms. Although ruptured distal MCA aneurysms are thought to be rare as sources of aneurysmal SAH, the incidence of ruptured distal MCA aneurysm was 9.4% of all ruptured MCA aneurysms in our study. Ruptured distal MCA aneurysms should be considered as sources of aneurysmal SAH without intracerebral hematoma., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose: To compare the function of retinal ganglion cells (RGCs) using the photopic negative response (PhNR) in patients who had undergone indocyaine green (ICG)-assisted, brilliant blue G (BBG)-assisted, or triamcinolone acetonide (TA)-assisted internal limiting membrane (ILM) peeling during macular hole (MH) surgery., Methods: Forty-eight eyes of 48 patients with a macular hole were randomly divided into those undergoing ICG-assisted, BBG-assisted, or TA-assisted vitrectomy (n = 16 for each group). Full-field cone ERGs were recorded before and 1, 3, 6, 9, and 12 months postoperatively. The amplitudes and implicit times of the a-waves and b-waves and the amplitudes of the oscillatory potentials (OPs) and PhNRs were measured. The mean deviations (MDs) of standard automated perimetry and the best-corrected visual acuity (BCVA) were measured. The circumferential retinal nerve fiber layer (RNFL) thickness was evaluated by SD-OCT., Results: All macular holes were closed with a significant improvement of the BCVA and MD without differences among the groups. There was no significant difference between the preoperative and postoperative RNFL thickness. The implicit times of the a-waves and b-waves were significantly prolonged, and the ΣOPs amplitude was significantly decreased postoperatively in all groups. These ERG changes were not significantly different among the groups. The postoperative PhNR amplitudes were significantly lower in the ICG group than in the BBG or TA group., Conclusions: The results indicate that the PhNR may detect subclinical impairments of RGCs caused by the possible toxic effect of ICG. This finding adds to the data that BBG and TA may be safer than ICG for use during MH surgery.

Purpose. To compare the postoperative changes of the retinal nerve fiber layer (RNFL) thickness in patients with macular holes (MHs) treated with vitrectomy with indocyanine green- (ICG-), brilliant blue G- (BBG-), or triamcinolone acetonide- (TA-)assisted internal limiting membrane (ILM) peeling. Methods. Sixty-one eyes of 61 consecutive patients with MHs were studied. Each eye was randomly selected to undergo either ICG- (n = 18), BBG- (n = 21), or TA-assisted (n = 22) ILM peeling. The circumferential retinal nerve fiber layer (RNFL) thickness was determined by spectral-domain optical coherence tomography (SD-OCT) before and 1, 3, 6, 9, and 12 months postoperatively. The mean overall and the sectoral thicknesses of the RNFL were obtained for each group. Results. A transient increase of the RNFL thickness was seen in the mean overall and sectoral thicknesses except for the nasal/inferior sector at 1 month after surgery for the three groups. Then, the thickness gradually decreased and returned to the baseline level in all sectors except for the nasal/inferior sector. The differences in the RNFL thickness among the groups were not significant for at least 12 months postoperatively. Conclusions. The degree of change of the RNFL thickness was not significantly related to the type of vital stain used during MH surgery.

Camptothecin (CPT) is an anti-tumor natural product that forms a ternary complex with topoisomerase I (top I) and DNA (CPT-top I-DNA). In this study, we identified the direct interaction between CPT and human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) using the T7 phage display technology. On an avidin-agarose bead pull down assay, hnRNP A1 protein was selectively pulled down in the presence of C20-biotinylated CPT derivative (CPT-20-B) both in vitro and in vivo. The interaction was also confirmed by an analysis on a quartz-crystal microbalance (QCM) device, yielding a K(D) value of 82.7 nM. A surface plasmon resonance (SPR) analysis revealed that CPT inhibits the binding of hnRNP A1 to top I (K(D): 260 nM) in a non-competitive manner. Moreover, an in vivo drug evaluation assay using Drosophila melanogaster showed that the knockout of the hnRNP A1 homolog Hrb87F gene showed high susceptibility against 5-50 μM of CPT as compared to a wild-type strain. Such susceptibility was specific for CPT and not observed after treatment with other cytotoxic drugs. Collectively, our data suggests that CPT directly binds to hnRNP A1 and non-competitively inhibits the hnRNP A1/top I interaction in vivo. The knockout strain loses the hnRNP A1 homolog as a both CPT-binding partner and naïve brakes of top I, which enhances the formation of the CPT-top I-DNA ternary complexes and subsequently sensitizes the growth inhibitory effect of CPT in D. melanogaster., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The purpose of the present study was to establish a rat bipedal walking model to examine the effects of bipedal walking on the central nervous system by training rats to perform bipedal walking over a period of 3 months. The characteristics of bipedal walking were investigated using kinematic and electromyographic methods in established bipedal walking models. Stable bipedal walking was achieved in rats by training them to stand with an upright posture and to walk with the hindlimbs using bipedal-walking training equipment to obtain a water reward. A stable head position in the rat bipedal walking model was attained primarily by closing the swing-phase period with a large angular change in the hip, knee, and ankle joints. The EMG burst pattern of the knee extensor (m. rectus femoris) and the erector muscle of the spine (m. longissimus) during bipedal walking was similar to that during quadrupedal walking in rats. We established two bipedal walking models using normal and forelimb-amputated rats. Comparative studies of these two bipedal walking models are expected to provide the information about the influence of forelimb movements on neuronal control of bipedal walking.

Purpose: To determine the clinical importance of the photopic negative response (PhNR) of the focal electoretinogram (fERG) for diagnosing glaucoma., Methods: Fifty-nine eyes of 38 patients with open-angle glaucoma (OAG), 12 glaucoma suspects, and 32 eyes of 32 normal controls were studied. The fERGs were elicited by a 15 degrees stimulus spot on the macula region, the supero-temporal, and infero-temporal regions of the macula. The mean of the visual sensitivity was measured by standard automated perimetry (SAP). The optimal cutoff amplitudes of the focal PhNR and ratios of the focal PhNR/b-wave amplitudes that discriminated glaucomatous eyes from normal eyes were obtained from the receiver operating characteristic curves., Results: The amplitudes of the PhNR were significantly smaller in patients with OAG than in normal controls (P < 0.00001). A curvilinear relationship was found between the mean sensitivity (dB, on log scale) and the PhNR amplitude, and between the mean sensitivity and the PhNR/b-wave amplitude ratio. After converting the mean sensitivity from a logarithmic to a linear scale, the amplitude of the PhNR and PhNR/b-wave ratio were linearly correlated with the SAP-determined visual sensitivity in all retinal areas (r = .428-0.544, P < 0.0001). When the optimal cutoff values were used, the sensitivity and specificity of the PhNR/b-wave amplitude ratio were 98.3% and 90.1%, respectively., Conclusions: The reduction of the focal PhNR amplitude was associated with a local decrease in the retinal sensitivity in OAG. The high sensitivity and specificity suggest that the focal PhNR can be used to detect functional loss in OAG.

Purpose: To investigate whether there is a significant correlation between the photopic negative response (PhNR) of the electroretinogram (ERG) and retinal nerve fiber layer thickness and optic disc topography in glaucomatous eyes., Methods: Ninety-nine eyes of 53 patients with open-angle glaucoma (OAG) and 30 eyes of 28 normal volunteers were studied. Photopic ERGs were elicited by red stimuli (644 nm, 1600 cd/m(2)) on a blue background (470 nm, 40 cd/m(2)). The mean deviation (MD) of the visual field was obtained by static visual field analyses. The topography of the optic nerve head was determined by confocal scanning laser ophthalmoscopy. The retinal nerve fiber layer thickness (RNFLT) around the optic nerve head was measured with a scanning laser polarimeter., Results: The amplitude of the PhNR and the PhNR/b-wave ratio decreased with an increase in visual field defects. The logarithmic values of the PhNR amplitude and PhNR/b-wave amplitude ratio were significantly correlated with the MD better than the linear values. The PhNR amplitude and PhNR/b-wave amplitude ratio were significantly correlated with the RNFLT and the rim area of the optic disc and with the cup/disc area ratio. These correlations were higher when expressed linearly than when stated logarithmically. The sensitivity and specificity were 77% and 90% for the PhNR amplitude and 70% and 87% for the PhNR/b-wave amplitude ratio when the optimal cutoff values were used. Although the a-wave amplitude correlated with the MD, the a-wave amplitudes of most of the patients fell within the normal range. The correlation between the b-wave amplitude and MD was not significant., Conclusions: The PhNR amplitudes correlate with the decrease in function and morphology of retinal neurons in eyes with OAG. The linear relationship between the PhNR and the structural parameters indicates that inner retinal function declines proportionately with neural loss in eyes with glaucoma.

Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 influence neuronal migration and have been identified in nasal regions. Gonadotropin releasing hormone-1 (GnRH-1) neurons migrate from nasal regions into the developing forebrain, where postnatally they control reproduction. This study examined the role of SDF-1/CXCR4 in development of the GnRH-1/olfactory systems. Migrating GnRH-1 neurons were CXCR4 immunopositive as were the fibers along which they migrate. SDF-1 transcripts were detected in olfactory epithelium and vomeronasal organ, while SDF-1 immunoreactivity highlighted the GnRH-1 migratory pathway. CXCR4-deficient mice showed a decrease in GnRH-1 cells at the nasal forebrain junction and in brain, but the overall migratory pathway remained intact, no ectopic GnRH-1 cells were detected and olfactory axons reached the olfactory bulb. To further characterize the influence of SDF-1/CXCR4 in the GnRH-1 system, nasal explants were used. CXCR4 expression in vitro was similar to that in vivo. SDF-1 was detected in a dorsal midline cell cluster as well as in migrating GnRH-1 cells. Treatment of explants with bicyclam AMD3100, a CXCR4 antagonist, attenuated GnRH-1 neuronal migration and sensory axon outgrowth. Moreover, the number of GnRH-1 neurons in the explant periphery was reduced. The effects were blocked by coincubation with SDF-1. Removal of midline SDF-1 cells did not alter directional outgrowth of olfactory axons. These results indicate that SDF-1/CXCR4 signaling in not necessary for olfactory axon guidance but rather influences sensory axon extension and GnRH-1 neuronal migration, and maintains GnRH-1 neuronal expression as the cells move away from nasal pit regions.

Unlabelled: Effect of milk basic protein on bone metabolism in healthy young women., Introduction: Milk has more beneficial effects on bone health than other food sources. Recent in vitro and in vivo studies have shown that milk whey protein, especially its basic protein fraction (milk basic protein, MBP), contains several components capable of promoting bone formation and inhibiting bone resorption. The object of this study was to examine the effect of MBP on the bone mineral density and bone metabolism of healthy young women., Methods: Thirty-five healthy young women were randomly assigned to treatment with either placebo or MBP (40 mg per day) for 6 months. The bone mineral density (BMD) of the lumbar vertebrae L2-L4 of each subject was measured by dual-energy X-ray absorptiometry (DXA) at 0 and 6 months of treatment. Serum and urine indexes of bone metabolism were measured at 0, 3 and 6 months. All subjects completed the study in accordance with the protocol., Results: The mean rate of gain of lumbar BMD in the MBP group (1.57%) was significantly higher than in the placebo group (0.13%, P=0.042). When compared with the placebo group, urinary cross-linked N-telopeptides of type-I collagen (NTx) were significantly decreased, and serum osteocalcin was significantly increased in the MBP group at 6 months., Conclusion: These results suggested that MBP supplementation was effective in increasing BMD in young women and that this increase in BMD may be primarily mediated through the promotion of bone formation and inhibition of bone resorption by MBP supplementation.

"Mainichi Hone Kea MBP" is a foods for specified health uses (FOSHU) product. It has been approved to write the next claim, "This product is suitable for those who care about bone health, because it contains MBP that has an effect to increase bone density", on this product. MBP is a complex of protein with a basic isoelectric point in milk protein. It has been shown that MBP promotes bone formation, suppresses bone resorption, increases bone mineral density, and increases bone strength in in vitro and in vivo studies. And, we also examined the effect of MBP in healthy adult women from twenties to fifties. A beverage containing MBP or a placebo beverage without MBP was given to volunteers for six months. The percentage increase of radial bone density was significantly higher in the MBP group than in the placebo group. This result has confirmed that MBP contributes to increased bone density in humans. To sum up, we believe that MAINICHI HONE KEA MBP, which contains MBP that has an effect to increase bone density, contributes to bone health.

Reasons for Performing Study: In laboratory animals, man and cell culture experiments, milk basic protein was reported to suppress bone resorption and promote bone formation. However, no studies in horses have previously examined the effect of milk basic protein., Objectives: To evaluate the effect of milk basic protein supplementation on bone metabolism in young Thoroughbred horses in training., Methods: Twenty 2-year-old horses in training were used for 90 days in this study. The treatment group was fed a basal diet with 1 g of milk basic protein and the control group a basal diet only. Blood samples were collected on Days 0, 45 and 90 to determine serum calcium (Ca) and biochemical markers of bone metabolism. Radiographs were taken at the start and end of the study to determine radiographic bone aluminium equivalence (RBAE)., Results: Serum osteocalcin (OC) was significantly higher at Day 45 after the beginning of the study in the treatment group compared to that in the control group. The treatment group showed a greater increase in the total RBAE change at the end of this study compared to that in the control group. However, there were no significant differences in serum Ca and carboxy-terminal telopeptide of type I collagen (ICTP) between groups., Conclusions and Potential Relevance: These findings provide preliminary evidence that milk basic protein has an effect on bone formation in 2-year-old Thoroughbred horses in training. However, further studies in larger groups of horses are now required to substantiate our findings.

Alfacarcidol and calcitriol are widely used to treat secondary hyperparathyroidism associated with chronic renal failure, but it is often not possible to administer doses high enough to sufficiently inhibit parathyroid hormones because of the risk of hypercalcemia and hyperphosphatemia. We administered falecalcitriol (Hornel) Tablets) to patients with poorly controlled secondary hyperparathyroidism. The usefulness of falecalcitriol was demonstrated by the fact that control of intact-PTH was maintained for up to 24 months without a clear increase in serum Ca x serum inorganic phosphorus (iP), iP, and ALP levels.

Milk has more beneficial effects on bone health than other food sources. Recent in vitro and in vivo studies have shown that milk whey protein, especially its basic protein fraction (milk basic protein, MBP), contains several components capable of promoting bone formation and inhibiting bone resorption. The object of this study was to examine the effect of MBP on the bone metabolism of healthy menopausal women. Thirty-two healthy menopausal women were randomly assigned to treatment with either placebo or MBP (40 mg per day) for 6 months. The bone mineral density (BMD) of the lumbar vertebrae L2-L4 of each subject was measured by dual-energy X-ray absorptiometry (DXA) at 0 and 6 months of treatment. Serum and urine indices of bone metabolism were measured at 0, 3 and 6 months. Twenty-seven subjects who completed the study in accordance with the protocol were included in the analysis. The mean rate of gain of lumbar BMD in the MBP group (1.21%) was significantly higher than in the placebo group (-0.66%, P=0.046). When compared with the placebo group, urinary cross-linked N-telopeptides of type-I collagen (NTx) were significantly decreased in the MBP group at 6 months, but no significant difference in serum osteocalcin was observed between the two groups. The urinary NTx excretion was found to be related to serum osteocalcin in the MBP group at 3 and 6 months, indicating that MBP maintained the balance of bone remodeling. These results suggested that MBP supplementation was effective in preventing bone loss in menopausal women and that this improvement in BMD may be primarily mediated through the inhibition of bone resorption while maintaining the balance of bone remodeling by MBP supplementation.

Migration of gonadotropin-releasing hormone-1 (GnRH-1) neurons from the nasal placode into the central nervous system occurs in all vertebrates. This study characterizes the expression of L- and N-type voltage-gated calcium channels (VGCCs) in migrating GnRH-1 neurons in mice. Class C (L-type) and class B (N-type) VGCGs were detected in GnRH-1 cells and cells in the olfactory and vomeronasal epithelium during prenatal development. This expression pattern was mimicked in a nasal explant model known to retain many characteristics of GnRH-1 development in vivo. Using this in vitro system, perturbation studies were performed to elucidate the role of VGCCs in GnRH-1 neuronal development. This report shows that olfactory axon outgrowth and GnRH-1 neuronal migration are attenuated when nasal explants are grown in calcium-free media, and that this effect is temporally restricted to an early developmental period. Blockade of either the L- or the N-type channel did not alter GnRH-1 cell number or overall olfactory axon outgrowth. However, blockade of N-type channels altered the distribution of GnRH-1 neurons in the periphery of the nasal explants. In these explants, more GnRH-1 neurons were located proximal to, and fewer GnRH-1 neurons distal to, the main tissue mass, suggesting a general decrease in the rate of GnRH-1 neuronal migration. These results indicate that extracellular calcium is required for initiating GnRH-1 neuronal migration and that these events are partially dependent on N-type VGCC signals.

Starch and dextrin were phosphorylated by dry-heating in the presence of phosphate, and their properties were examined. The phosphorylation of starch was accelerated with decreasing moisture, rising incubation temperature and prolongation of the incubation period. However, a rise in incubation temperature, and lengthened incubation period caused increased browning and degradation. A decrease in the pH from 5.5 to 3.0 resulted in a modest increase of phosphorylation, but also marked browning and degradation. When potato starch was phosphorylated at 140 degrees C and pH 5.5 for 24 h, its phosphorus content was increased up to 3.47%, with fewer side reactions. Phosphorlylated starch and dextrin had calcium phosphate-solubilizing abitity. Phosphorylated dextrin with 2.42% phosphorus had about half the calcium phosphate-solubilizing ability of casein phosphopeptide (CPP). The possibility of substituting of phosphorylated starch and dextrin for CPP as a calcium phosphate-absorption enhancing material is discussed herein.

A cystein protease inhibitor was identified in the basic fraction of bovine milk. We have reported in our previous study that the milk basic protein (MBP) fraction suppressed osteoclast-mediated bone resorption in vitro. Since osteoclasts secreted cystein protease to digest collagen in the bone matrix, we identified the cystein protease inhibitor in MBP. A 12-kDa inhibitor was purified from MBP by papain affinity gel chromatography and subsequent Hi-Load Superdex 75 gel filtration chromatography. The N-terminal sequence of the 18 amino acid residues of the inhibitor corresponded to bovine cystatin C. The 12-kDa cystein protease inhibitor in MBP therefore seemed to be cystatin C. Purified cystatin suppressed bone resorption with the use of isolated osteoclasts in vitro. Cystatin in MBP is suggested as one of the factors inhibiting bone resorption.

We investigated the calcium bioavailability of milk calcium, taken with or without cheese. Twenty-four 6-week-old male rats for a meal-feeding experiment were trained to consume an AIN-76 diet within 2 h (2 times per day) for 2 weeks. The rats were then divided into three experimental groups, each fed 2 types of experimental diets: Control group, Cheese group, and Ca-Cheese group. The rats were each alternately given 2 types of experimental diets at 2-h meal-feeding for 31 days. The breaking force and energy of the femur in the Ca-Cheese group were significantly higher than in the control group. The bone mineral density (BMD) of the lumbar spine and the femur in the Ca-Cheese group was also significantly higher than in the other two groups. These results indicate that milk calcium taken with cheese increases bone strength and BMD efficiently, results that may be useful for the prevention of osteoporosis.

We prepared a novel type of milk calcium from milk whey, and evaluated its calcium bioavailability as compared with conventional industrial milk calcium obtained from milk whey, which has been reported to be an excellent calcium source. An X-ray diffraction analysis showed that the chemical form of conventional industrial milk calcium from milk whey through the neutralization and precipitation method (NP-MCa) is hydroxyapatite type, and that of a novel type of milk calcium obtained using the ultrafiltration method (UF-MCa) is different from hydroxyapatite. Growing rats were fed either NP-MCa or UF-MCa as the source of calcium for 33 d. The calcium absorption rate in the UF-MCa diet group was significantly higher than that in the NP-MCa diet group. The calcium content of the femur in the UF-MCa diet group was significantly higher than that in the NP-MCa diet group. The breaking force of the excised femur in the UF-MCa diet group was also significantly higher than that in the NP-MCa diet group. The serum calcium level, alkaline phosphatase activity and calcitonin level were not significantly different between the experimental groups, but the PTH level of the serum in the UF-MCa diet group was significantly lower than that in the NP-MCa diet group. These results indicate that the calcium bioavailability of the novel type of milk calcium (UF-MCa) is higher than that of the hydroxyapatite type of milk calcium (NP-MCa).

N-syndecan, a membrane-bound heparan sulphate proteoglycan, is abundantly present in the developing nervous system and thought to play important roles in the neurite outgrowth. In the present study, we examined the distribution of N-syndecan in the migratory route from the rat olfactory placode using immunohistochemistry and in situ hybridization. At embryonic day 15, both heparan sulphate and N-syndecan immunoreactivities were localized in and around the migrating cell clusters, which contained luteinizing hormone-releasing hormone (LHRH) and calbindin D-28k. Immunoreactivity for other glycosaminoglycan chains, such as chondroitin and keratan sulphate, and core proteins of the chondroitin sulphate proteoglycan, neurocan and phosphacan, were barely detected in the migratory pathway from the olfactory placode. By in situ hybridization histochemistry, N-syndecan mRNA was localized in virtually all of migrating neurons as well as in cells of the olfactory epithelium and the vomeronasal organ. N-syndecan immunoreactivity surrounded cells migrating along the vomeronasal nerves that were immunoreactive for neural cell adhesion molecules, NCAM, L1 and TAG-1. Considering that NCAM is implicated in the migratory process of LHRH neurons and specifically binds to heparan sulphate, it is likely that a heterophilic interaction between NCAM and N-syndecan participates in the neuronal migration from the rat olfactory placode.

We studied the effects of daily intake of milk basic protein (MBP) on radial bone mineral density (BMD) in healthy adult women. Thirty-three healthy women were randomly assigned to a 6-month trial with either placebo or MBP (40 mg per day). The radial BMD of each volunteer was measured at the beginning of and at six months after the trial. The mean BMD value at the 6th month in the MBP group increased significantly at both 1/6 and 1/10 portion from the distal end of the radius, whereas that in the control group did not. The BMD gain of each volunteer in the MBP group was significantly higher than that in the placebo group. Thus a daily MBP supplementation of 40 mg in healthy adult women can significantly increase radial BMD.

The change of the toxicity of chlorinated water after chlorine injection was examined. For the measurement of toxicity, chromosomal aberration test and transforming test were carried out as indexes to initiating activity and to promoting activity in the carcinogenesis process, respectively. Activity inducing chromosomal aberrations of chlorinated humic acid gradually decreased with time after chlorination. In contrast, activity inducing transformations measured by the two-stage assay gradually increased. Thus, the toxicity that decreases or increases is present in chlorinated water. Furthermore, activity inducing transformations measured by the non-two-stage assay gradually decreased. This direction of change was reverse to that of activity inducing transformations by the two-stage assay and consistent with that of activity inducing chromosomal aberrations. It is speculated that the main reason of decreasing activity inducing transformations by the non-two-stage assay is because initiating activity detected as activity inducing chromosomal aberrations in chlorinated water decreases drastically. Directions of changes of total organic halogen and carbonyl group were qualitatively consistent with that of activity inducing chromosomal aberrations. Directions of changes of chloroform and dichloroacetic acid were qualitatively consistent with that of activity inducing transformations by the two-stage assay. Findings of this study suggest that further research is necessary to compare carcinogenicity of tap water near water purification plant and distant tap water.

The spatiotemporal localization of calbindin D-28k (Calb), a calcium-binding protein, was examined immunohistochemically in the developing rat olfactory system with special reference to cell migration from the olfactory placode. Calb immunoreactivity was first detected at embryonic day 12 (E12) in a few cells just outside the olfactory epithelium, and at E13, Calb-immunoreactive cells were found scattered in the laminin-rich mesenchyme. By E14, Calb-immunoreactive cells had increased in number and were seen along the entire migratory route between the vomeronasal organ, a derivative of the medial olfactory pit, and the ventromedial surface of the telencephalic vesicle. Calb neurones were not seen in the olfactory epithelium, a derivative of the lateral olfactory pit. Although the distribution pattern of Calb-immunoreactive cells was similar to that of luteinizing hormone releasing hormone (LHRH)-producing neurones, which are known to originate in the vomeronasal organ and migrate into the forebrain, Calb and LHRH immunoreactivities were contained in separate neuronal populations. Calb-immunoreactive cells were localized along the vomeronasal nerves, identified by labelling the vomeronasal organ with the lipophilic dye, DiI, and strongly immunoreactive for neural cell adhesion molecule (NCAM). These data strongly suggest that, in addition to LHRH neurones, the rat vomeronasal organ generates Calb-immunoreactive neurones which migrate along the vomeronasal nerves to enter the forebrain. The final fate and functional importance of these cells remains to be determined.

Milk contains several components effective for bone health. In the previous in vitro and in vivo studies, we have shown that milk whey protein, especially its basic protein fraction (milk basic protein [MBP]), promoted bone formation and suppressed bone resorption. This present study examines the effect of MBP on the biochemical markers of bone metabolism in healthy adult men. Experimental beverages containing MBP (300 mg of MBP a day) were given to 30 normal healthy adult men for 16 days. The serum osteocalcin concentration had increased significantly after 16 days of ingesting the experimental beverage containing MBP. Urinary cross-linked N-teleopeptides of type-I collagen (NTx) excretion had decreased significantly after 16 days of ingesting MBP. The urinary NTx excretion was related to the serum osteocalcin concentration after 16 days of ingestion. These results suggest that MBP promoted bone formation and suppressed bone resorption, while maintaining the balance of bone remodeling.

Milk has more beneficial effects on bone health compared to other food sources. Recent in vitro and in vivo studies showed that milk whey protein, especially its basic protein fraction, contains several components capable of both promoting bone formation and inhibiting bone resorption. However, the effects of milk basic protein (MBP) on bone metabolism of humans are not known. The object of this study was to examine the effects of MBP on bone metabolism of healthy adult women. Thirty-three normal healthy women were randomly assigned to treatment with either placebo or MBP (40 mg per day) for six months. The bone mineral density (BMD) of the left calcaneus of each subject was measured at the beginning of the study and after six months of treatment, by dual-energy x-ray absorptiometry. Serum and urine indices of bone metabolism were measured at the base line, three-month intervals, and the end of the study. Daily intake of nutrients was monitored by a three-day food record made at three and six months. The mean (+/- SD) rate of left calcaneus BMD gain of women in the MBP group (3.42 +/- 2.05%) was significantly higher than that of women in the placebo group (2.01 +/- 1.75%, P = 0.042). As compared with the placebo group, urinary cross-linked N-teleopeptides of type-I collagen/creatinine and deoxypyridinoline/creatinine were significantly decreased in the MBP group (p < 0.05), while no significant differences between the two groups were observed in serum osteocalcin and bone-specific alkaline phosphatase concentrations. A daily MBP supplementation of 40 mg in healthy adult women can significantly increase their BMD independent of dietary intake of minerals and vitamins. This increase in BMD might be primarily mediated through inhibition of osteoclast-mediated bone resorption by the MBP supplementation.

Milk is recommended as an excellent calcium source for bone health. Moreover, milk is considered to contain other components effective for bone health. In our previous studies, using an unfractionated bone cell culture system, we found that milk whey protein, especially its basic fraction (milk basic protein [MBP]), suppressed bone resorption. In this present study, we investigated whether MBP could prevent bone loss in aged ovariectomized rats. Twenty-one 51-week-old female Sprague-Dawley rats were ovariectomized (ovx), and another seven rats received a sham operation (sham). After a 4-week recovery period, the ovx rats were separated into three groups, and they were then fed a control diet, a 0.01% MBP diet (0. 01% casein of the control diet replaced with MBP), or a 0.1% MBP diet for 17 weeks. The sham rats were fed the control diet. Bone mineral density (BMD) of the femur was measured by dual-energy X-ray absorptiometry in vivo. The BMD in the ovx-control group noticeably decreased during the experimental period in comparison with that in the sham group. However, the BMD in the OVX-0.1% MBP group was significantly higher than that in ovx-control group at weeks 12 and 16 (p < 0.05). After the 17-week feeding period, the breaking energy of the excised femur of all groups was determined by use of a three-point bending rheolometer. The breaking energy in the ovx-control group was significantly lower than that in the sham group (p < 0.05). However, the breaking energy in the ovx-0.1% MBP group was significantly higher than that of the ovx-control group (p < 0.05). Urinary deoxypyridinoline (D-Pyr) level of the ovx-control group was higher than that of the sham group, whereas the level of D-Pyr excretion in the ovx-0.01% MBP and ovx-0.1% MBP groups was significantly lower than that of the ovx-control group (p < 0.05). These results suggest that MBP suppresses the osteoclast-mediated bone resorption and prevents bone loss caused by ovariectomy. Moreover, we performed an in vitro study using isolated osteoclasts from rabbit bone to investigate the possible mechanism. MBP dose-dependently suppressed the number of pits formed by these osteoclasts. This result indicates that MBP suppresses bone resorption by its direct effects on osteoclasts. To our knowledge, this study provides the first evidence that MBP directly suppresses osteoclast-mediated bone resorption, resulting in the prevention of the bone loss that occurs in ovx rats.

Milk calcium salt solution was prepared by the following procedures using casein phosphopeptides (CPP). To CPP solution, 1 M citric acid, 1 M CaCl2 and 1 M K2HPO4 were added with stirring, while adjusting the pH to 6.7. The prepared solution was left for 12 hr at 25 degrees C and then used for subsequent experiments, or lyophilized. The concentrations of organic phosphate of CPP, calcium, inorganic phosphate, and citrate in the typical milk salt solution were 7, 30, 22, and 10 mM, respectively, which were close to those in bovine milk. The lyophilized sample was easily dissolved in water. No crystal structure of hydroxyapatite was shown in the lyophilized milk calcium salts by X-ray diffraction analysis, although the pattern of KCl crystal was observed. The X-ray diffraction pattern of commercial whey mineral, which was prepared by precipitation at alkaline pH from rennet whey, was similar to that of hydroxyapatite. It was confirmed by high-performance gel chromatographic analysis that the form of calcium phosphate in the milk calcium salts was similar to that of casein micelles.

Objective: We describe an easy and useful method for treating hemifacial spasm related to the vertebral artery., Methods: The technique entails the manufacture of a dural belt harvested from the cerebellar convexity dura and a dural bridge made at the petrous dura combined with the use of an aneurysm clip. The dural belt holds the vertebral artery and is anchored to the dural bridge by fixation with an aneurysm clip after the vertebral artery is transposed to an appropriate position., Results: The technique proved to be safe and effective in a series of six patients with hemifacial spasm who were followed up for a period of 2 months to more than 10 years after surgery. All patients were affected on the left side. Multiple offending arteries were present in three cases. Hemifacial spasm completely disappeared in all patients., Conclusion: This method represents a feasible option for the treatment of hemifacial spasm caused by a tortuous, elongated, or enlarged vertebral artery.

We evaluated the bioavailability of two types of calcium from milk in two experiments. One was a micellar calcium phosphate-phosphopeptide (MCP-PP) complex in which the chemical form was similar to the original form of milk, and the other was a commercial whey calcium in which the chemical form was different from that of milk. In experiment 1, the calcium absorption, bone mineral density, and bone strength were examined when growing female rats were fed either MCP-PP complex or whey calcium as the sole source of calcium for 46 d. In experiment 2, the calcium solubility in the small intestine was measured when female rats were meal-fed either MCP-PP complex or whey calcium. The apparent calcium absorption rate in both groups decreased time-dependently during the experimental period, but the time-dependent change in the apparent calcium absorption rate was statistically different. It decreased more slowly in rats fed the MCP-PP diet than in rats fed the whey calcium diet. The bone mineral density of the femur in rats fed the MCP-PP diet was significantly higher than that of the rats fed the whey calcium diet. The bone strength (breaking force and energy) of the femur in rats fed the MCP-PP diet was higher than in the rats fed the whey calcium diet. The amount of soluble calcium in the small intestinal contents in rats at 2.5 h after ingestion of the MCP-PP diet was approximately three times higher than that found in rats fed the whey calcium diet. These results indicate that the calcium bioavailability of MCP-PP complex is higher than that of whey calcium, and this difference is due in part to the solubility in the intestine.

Micellar calcium phosphate (MCP) in bovine milk was separated as the complex with casein phosphopeptide (CPP) by the following procedures. Rennet curd obtained from skim milk was suspended in water, the pH was adjusted to 4.6, and the suspension was centrifuged at 1,000 x g. CPP was separated from the precipitated casein by tryptic hydrolysis and ethanol precipitation. The supernatant, which contained calcium and inorganic phosphate liberated from casein micelles by acidification, and CPP were mixed; the pH was adjusted to 6.7; and then the solution was lyophilized. From 1 L of skim milk, 3.16 g of the MCP-CPP complex was obtained. The dried MCP-CPP complex was easily dissolved in water and contained 12.7% calcium, 0.3% magnesium, 3.4% inorganic phosphorous, and 2.2% organic phosphorous. No crystal structure of hydroxyapatite was shown in the MCP-CPP complex by the X-ray diffraction analysis, although the pattern of NaCl crystal was observed. The X-ray diffraction pattern of commercial whey mineral, which was prepared by precipitation at alkaline pH from rennet whey, was similar to that of hydroxyapatite. It was confirmed by high-performance gel chromatographic analysis that the form of calcium phosphate in the MCP-CPP complex was similar to that of casein micelles. The MCP-CPP complex was also separated from commercial rennet casein. The method for the separation of MCP-CPP complex described above can be applied to the large-scale preparation.