Improved seizure liability detection by combining rat hippocampal brain slice electrophysiology with in vivo behavior observation following intracerebroventricular drug administration.

An adverse effect of drug candidates, seizure is a serious issue in drug development. Improving evaluation systems for seizure liability is crucial for selecting good candidates. Firstly, in vitro electrophysiological measurement by a multielectrode array system in rat hippocampal brain slices was employed to confirm an increase in electrically evoked population spike (PS) area, the occurrence of multiple population spikes (MPSs), and thereby the seizure liability of five positive control chemicals: picrotoxin, 4-aminopyridine, pentylenetetrazole, penicillin G, and chlorpromazine. Aspirin, a negative control, did not affect PS area or generate MPSs. Furthermore, baclofen, an anticonvulsant drug, decreased PS area and inhibited the increase in PS area or occurrence of MPSs induced by picrotoxin. A comparative study of seizure liability among carbapenem antibiotics revealed that tienam > carbenin > omegacin and finibax. Despite leading to a strong decrease in PS area, physostigmine, cisplatin, and paroxetine still produced MPSs. Therefore, the increase in PS area or the occurrence of the MPS are considered significant evaluation parameters for seizure liability. In contrast, the in vitro electrophysiological measurement could not detect the seizure liability of diphenhydramine or fluvoxamine. A follow-up study of in vivo mouse behavioral change induced by intracerebroventricular administration of these drugs clearly detected convulsions. The in vitro electrophysiological study using hippocampal brain slices combined with in vivo behavior observation study of drug candidates administered by intracerebroventricular injection can implement to assess the seizure liability of even small amounts, especially in the early stages of drug development.

Candida dubliniensis in Japanese Oral Microbiota: A Cross-Sectional Study of Six Geographic Regions in Japan.

INTRODUCTION: Candida dubliniensis was reclassified from the C. albicans genotype D, and reports show its frequent detection in HIV-positive individuals and easy acquisition of antifungal drug resistance. However, the oral carriage rate in healthy people and contribution to candidiasis in Japan is unclear. METHODS: We conducted a cross-sectional survey of the C. dubliniensis carriage rate, performed genotyping and tested antifungal drug susceptibility and protease productivity. Specimens from 2432 Japanese subjects in six regions (1902 healthy individuals, 423 with candidiasis individuals, 107 HIV-positive individuals) were cultured using CHROMagarTMCandida, and the species was confirmed via 25S rDNA amplification and ITS sequences analyzed for genotyping. RESULTS: The C. dubliniensis carriage rate in healthy Japanese was low in the central mainland (0-15%) but high in the most northerly and southerly areas (30-40%). The distribution of these frequencies did not differ depending on age or disease (HIV-infection, candidiasis). Genotype I, previously identified in other countries, was most frequent in Japan, but novel genotypes were also observed. Six antifungal drugs showed higher susceptibility against C. albicans, but protease productivity was low. CONCLUSIONS: Oral C. dubliniensis has low pathogenicity with distribution properties attributed to geography and not dependent on age or disease status.

Cyb5r3 activation rescues secondary failure to sulfonylurea but not ß-cell dedifferentiation.

Diabetes mellitus is characterized by insulin resistance and ß-cell failure. The latter involves impaired insulin secretion and ß-cell dedifferentiation. Sulfonylurea (SU) is used to improve insulin secretion in diabetes, but it suffers from secondary failure. The relationship between SU secondary failure and ß-cell dedifferentiation has not been examined. Using a model of SU secondary failure, we have previously shown that functional loss of oxidoreductase Cyb5r3 mediates effects of SU failure through interactions with glucokinase. Here we demonstrate that SU failure is associated with partial ß-cell dedifferentiation. Cyb5r3 knockout mice show more pronounced ß-cell dedifferentiation and glucose intolerance after chronic SU administration, high-fat diet feeding, and during aging. A Cyb5r3 activator improves impaired insulin secretion caused by chronic SU treatment, but not ß-cell dedifferentiation. We conclude that chronic SU administration affects progression of ß-cell dedifferentiation and that Cyb5r3 activation reverses secondary failure to SU without restoring ß-cell dedifferentiation.

Essential oil components of turmeric inhibit hepatic lipidification and liver fibrosis in a diet-induced NASH model rats.

In this study, the fraction extracted from turmeric powder with 50% ethanol and fractionated with n-hexane were administered to diet-induced NASH model rats. NASH model was prepared with SD rats by feeding an originally designed choline-deficient, high-fat, high-fructose (HFF-CD) diet for 10 weeks. To the HFF-CD diet, hexane fraction and 50% ethanol fraction after hexane fractionation were added at 100 mg/kg body weight. 10 weeks later, blood samples and liver were collected for the following parameters: lipid weights, serum ALT, AST, TG, liver TG, TBARS levels, lipid metabolism-related gene expression and histopathological examination of the liver. As the results, the hexane fraction and 50% ethanol fraction showed a decrease in lipid weight, a decrease in hepatic TG, and activation of PPAR-α in the lipid metabolism-related gene test. These results suggest that the hexane fraction of turmeric has an inhibitory effect on fat accumulation in the liver by promoting lipid metabolism in NASH model rats.

Inclusive greenhouse gas budget assessment in turfs: From turf production to disposal of grass clippings.

Globally, greenhouse gas (GHG) reduction is a serious concern. To evaluate whether turfs serve as a GHG sink or source, GHG budget assessments for life cycle are required. However, previous studies have only focused on the use of turfs. To bridge these gaps in literature, this study investigated GHG (CO2, N2O, and CH4) emissions from the disposal of grass clippings and soil GHG fluxes in turfs. Additionally, GHG budgets in the turf production phase were assessed. Finally, inclusive GHG budgets from turf production to disposal of grass clippings for four turf uses (soccer stadium, golf course, office, and urban park) were assessed. Grass clippings were disposed in three forms (incineration, leaving as-is, and biochar). We found that GHG emissions from incineration and leaving 1 t-fresh weight (FW) of grass clippings were 0.711 and 0.207 t-CO2e, respectively. Contrastingly, the GHG emissions from the biochar yield from 1 t-FW of grass clippings were -0.200 t-CO2e. Further, annual soil GHG fluxes in newly established Zoysia and Kentucky bluegrass turfs were calculated at 0.067 and 0.040 tCO2eï½¥ha-1ï½¥yr-1, respectively. As the turf grass in production fields sequester large amounts of CO2, GHG budgets in turf production phase were estimated at approximately -20 t-CO2eï½¥ha-1ï½¥yr-1. Inclusive GHG budget assessment from turf production to disposal of grass clippings showed that turfs only in the urban parks served as a GHG sink and this ability was comparable to CO2 sequestration in forests. To enhance the ability of GHG sinks and to promote changes from a GHG source to GHG sink, our study revealed the importance of reduction of GHG emissions from energy and resource uses (especially fertilizers and gasoline) for turf management.

[Retracted] Proteasome inhibitor sensitizes oral squamous cell carcinoma cells to TRAIL­mediated apoptosis.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that, in Fig. 4 on p. 650, the same ß­actin bands had apparently been used to show the experimental effects of the proteasome inhibitor MG­132 on c­FLIP in HSC­2 cells in Fig. 4A, and the effects of MG­132 on IAPs in HSC­3 cells in Fig. 4B. In addition, for the fourth lane in the gel showing the effects of MG­132 on c­FLIP in HSC­3 cells, this should have been labelled as '+MG­132 / +TRAIL' (not as '­/­'). Upon contacting the authors in relation to this matter, they could only admit that errors had been made in the preparation of the figure; moreover, they no longer had access to the original data owing to the time that has elapsed since the publication of the paper, and it would be impossible for them to now repeat this experiment. After having considered this matter and in conjunction with a request made by the authors, the Editor of Oncology Reports has decided that this paper should be retracted from the publication. Both the Editor and the authors apologize to the readership for any inconvenience caused. [Oncology Reports 25: 645­652, 2011; DOI: 10.3892/or.2010.1127].

Health Literacy among Japanese College Students: Association with Healthy Lifestyle and Subjective Health Status.

The improvement of health literacy (HL) is a critical issue for college students who are in the transitional period to adulthood and are establishing their subsequent lifestyles. The present study aimed to evaluate the current state of HL among college students and to explore the factors that influence HL. Moreover, it investigated the relationship between HL and health conditions. For this study, the researchers conducted an online survey of college students. The questionnaire consisted of the Japanese version of the 47-item European Health Literacy Survey Questionnaire (HLS-EU-Q47), which is a self-assessment tool for HL that covers the major health issues of college students and health-related quality of life. The study analyzed 1049 valid responses. Based on the HLS-EU-Q47 total score, 85% of the participants exhibited problematic or unsatisfactory HL levels. Participants who reported high levels of healthy lifestyles obtained high HL scores. High levels of HL were associated with high levels of subjective health. Results from quantitative text analysis suggested that specific mindsets were correlated with high levels of competency in appraising health information among male students. In the future, educational intervention programs for college students need to be established to improve HL levels.

Cyb5r3-based mechanism and reversal of secondary failure to sulfonylurea in diabetes.

Sulfonylureas (SUs) are effective and affordable antidiabetic drugs. However, chronic use leads to secondary failure, limiting their utilization. Here, we identify cytochrome b5 reductase 3 (Cyb5r3) down-regulation as a mechanism of secondary SU failure and successfully reverse it. Chronic exposure to SU lowered Cyb5r3 abundance and reduced islet glucose utilization in mice in vivo and in ex vivo murine islets. Cyb5r3 ß cell-specific knockout mice phenocopied SU failure. Cyb5r3 engaged in a glucose-dependent interaction that stabilizes glucokinase (Gck) to maintain glucose utilization. Hence, Gck activators can circumvent Cyb5r3-dependent SU failure. A Cyb5r3 activator rescued secondary SU failure in mice in vivo and restored insulin secretion in ex vivo human islets. We conclude that Cyb5r3 is a key factor in the secondary failure to SU and a potential target for its prevention, which might rehabilitate SU use in diabetes.

Reflections on the state of diabetes research and prospects for treatment.

Research on the etiology and treatment of diabetes has made substantial progress. As a result, several new classes of anti-diabetic drugs have been introduced in clinical practice. Nonetheless, the number of patients achieving glycemic control targets has not increased for the past 20 years. Two areas of unmet medical need are the restoration of insulin sensitivity and the reversal of pancreatic beta cell failure. In this review, we integrate research advances in transcriptional regulation of insulin action and pathophysiology of beta cell dedifferentiation with their potential impact on prospects of a durable "cure" for patients suffering from type 2 diabetes.

The transcription factor ATF3 switches cell death from apoptosis to necroptosis in hepatic steatosis in male mice.

Hepatocellular death increases with hepatic steatosis aggravation, although its regulation remains unclear. Here we show that hepatic steatosis aggravation shifts the hepatocellular death mode from apoptosis to necroptosis, causing increased hepatocellular death. Our results reveal that the transcription factor ATF3 acts as a master regulator in this shift by inducing expression of RIPK3, a regulator of necroptosis. In severe hepatic steatosis, after partial hepatectomy, hepatic ATF3-deficient or -overexpressing mice display decreased or increased RIPK3 expression and necroptosis, respectively. In cultured hepatocytes, ATF3 changes TNFα-dependent cell death mode from apoptosis to necroptosis, as revealed by live-cell imaging. In non-alcoholic steatohepatitis (NASH) mice, hepatic ATF3 deficiency suppresses RIPK3 expression and hepatocellular death. In human NASH, hepatocellular damage is correlated with the frequency of hepatocytes expressing ATF3 or RIPK3, which overlap frequently. ATF3-dependent RIPK3 induction, causing a modal shift of hepatocellular death, can be a therapeutic target for steatosis-induced liver damage, including NASH.

Ultrafast lumbar spine MRI protocol using deep learning-based reconstruction: diagnostic equivalence to a conventional protocol.

OBJECTIVE: To evaluate the diagnostic equivalency between an ultrafast (1 min 53 s) lumbar MRI protocol using deep learning-based reconstruction and a conventional lumbar MRI protocol (12 min 31 s). MATERIALS AND METHODS: This study included 58 patients who underwent lumbar MRI using both conventional and ultrafast protocols, including sagittal T1-weighted, T2-weighted, short-TI inversion recovery, and axial T2-weighted sequences. Compared with the conventional protocol, the ultrafast protocol shortened the acquisition time to approximately one-sixth. To compensate for the decreased signal-to-noise ratio caused by the acceleration, deep learning-based reconstruction was applied. Three neuroradiologists graded degenerative changes and analyzed for presence of other pathologies. For the grading of degenerative changes, interprotocol intrareader agreement was assessed using kappa statics. Interchangeability between the two protocols was also tested by calculating the individual equivalence index between the intraprotocol interreader agreement and interprotocol interreader agreement. For the detection of other pathologies, interprotocol intrareader agreement was assessed. RESULTS: For the grading of degenerative changes, the kappa values for interprotocol intrareader agreement of all three readers ranged from 0.707 to 0.804, indicating substantial to almost perfect agreement. Except for foraminal stenosis and disc contour on axial images, the 95% confidence interval of the individual equivalence index was < 5%, indicating the two protocols were interchangeable. For the detection of other pathologies, the interprotocol intrareader agreement rates were > 98% for each individual pathology. CONCLUSIONS: Our proposed ultrafast lumbar spine MRI protocol provided almost equivalent diagnostic results to that of the conventional protocol, except for some degenerative changes.

Posteromedial vertical capsulotomy selectively expands the intraoperative extension gap in cruciate-retaining total knee arthroplasty.

PURPOSE: Effective soft-tissue balancing procedures for expanding the extension gap (EG) are needed in cases of gap mismatch in total knee arthroplasty (TKA). A posteromedial vertical capsulotomy (PMVC) is performed to restore mobility in a knee with a flexion contracture. The purpose of this study was to evaluate the effectiveness and safety of PMVC for intraoperative gap adjustment in cruciate-retaining TKA. METHODS: A total of 120 consecutive knees undergoing cruciate-retaining TKA for varus osteoarthritis were examined. The EG and flexion gap (FG) with a trial femoral component were measured using spacer blocks before and after PMVC. PMVC was performed when the first FG was larger than the first EG by > 2 mm. RESULTS: Sixty-five knees underwent PMVC, and the mean EG significantly increased by 2.4 mm (p < 0.001). This increase was significantly larger than that of the FG by 2.0 mm (p < 0.001). The preoperative extension range of motion (ROM) was negatively correlated with the EG change after PMVC (r = - 0.39, p = 0.001). A receiver operating characteristic (ROC) curve indicated a preoperative extension ROM cut-off of -10° for predicting PMVC (sensitivity 72.3%, specificity 56.4%). No associated complications were observed during a minimum 2-year follow-up period, and there was no difference in the postoperative Knee Society Score between the PMVC and non-PMVC groups. CONCLUSION: PMVC may be a useful soft-tissue treatment for gap adjustment with a selective EG expansion in TKA, especially in cases of a limited preoperative extension of - 10° or less. LEVEL OF EVIDENCE: Therapeutic study, level III.

Chemical induction of gut ß-like-cells by combined FoxO1/Notch inhibition as a glucose-lowering treatment for diabetes.

OBJECTIVE: Lifelong insulin replacement remains the mainstay of type 1 diabetes treatment. Genetic FoxO1 ablation promotes enteroendocrine cell (EECs) conversion into glucose-responsive ß-like cells. Here, we tested whether chemical FoxO1 inhibitors can generate ß-like gut cells. METHODS: We used Ngn3-or Villin-driven FoxO1 ablation to capture the distinctive developmental effects of FoxO1 on EEC pool. We combined FoxO1 ablation with Notch inhibition to enhance the expansion of EEC pool. We tested the ability of an orally available small molecule of FoxO1 inhibitor, Cpd10, to phenocopy genetic ablation of FoxO1. We evaluated the therapeutic impact of genetic ablation or chemical inhibition of FoxO1 on insulin-deficient diabetes in Ins2Akita/+ mice. RESULTS: Pan-intestinal epithelial FoxO1 ablation expanded the EEC pool, induced ß-like cells, and improved glucose tolerance in Ins2Akita/+ mice. This genetic effect was phenocopied by Cpd10. Cpd10 induced ß-like cells that released insulin in response to glucose in gut organoids, and this effect was enhanced by the Notch inhibitor, DBZ. In Ins2Akita/+ mice, a five-day course of either Cpd10 or DBZ induced intestinal insulin-immunoreactive ß-like cells, lowered glycemia, and increased plasma insulin levels without apparent adverse effects. CONCLUSION: These results provide proof of principle of gut cell conversion into ß-like cells by a small molecule FoxO1 inhibitor, paving the way for clinical applications.

Pharmacological conversion of gut epithelial cells into insulin-producing cells lowers glycemia in diabetic animals.

As a highly regenerative organ, the intestine is a promising source for cellular reprogramming for replacing lost pancreatic ß cells in diabetes. Gut enterochromaffin cells can be converted to insulin-producing cells by forkhead box O1 (FoxO1) ablation, but their numbers are limited. In this study, we report that insulin-immunoreactive cells with Paneth/goblet cell features are present in human fetal intestine. Accordingly, lineage-tracing experiments show that, upon genetic or pharmacologic FoxO1 ablation, the Paneth/goblet lineage can also undergo conversion to the insulin lineage. We designed a screening platform in gut organoids to accurately quantitate ß-like cell reprogramming and fine-tune a combination treatment to increase the efficiency of the conversion process in mice and human adult intestinal organoids. We identified a triple blockade of FOXO1, Notch, and TGF-ß that, when tested in insulin-deficient streptozotocin (STZ) or NOD diabetic animals, resulted in near normalization of glucose levels, associated with the generation of intestinal insulin-producing cells. The findings illustrate a therapeutic approach for replacing insulin treatment in diabetes.

Prevention of mastitis in multiparous dairy cows with a previous history of mastitis by oral feeding with probiotic Bacillus subtilis.

Mastitis is a very common inflammatory disease of the mammary gland of dairy cows, resulting in a reduction of milk production and quality. Probiotics may serve as an alternative to antibiotics to prevent mastitis, and the use of probiotics in this way may lessen the risk of antibiotic resistant bacteria developing. We investigated the effect of oral feeding of probiotic Bacillus subtilis (BS) C-3102 strain on the onset of mastitis in dairy cows with a previous history of mastitis. BS feeding significantly decreased the incidence of mastitis, the average number of medication days and the average number of days when milk was discarded, and maintained the mean SCC in milk at a level substantially lower than the control group. BS feeding was associated with lower levels of cortisol and TBARS and increased the proportion of CD4+ T cells and CD11c+ CD172ahigh dendritic cells in the blood by flow cytometry analysis. Parturition increased the migrating frequency of granulocytes toward a milk chemoattractant cyclophilin A in the control cows, however, this was reduced by BS feeding, possibly indicating a decreased sensitivity of peripheral granulocytes to cyclophilin A. These results reveal that B. subtilis C-3102 has potential as a probiotic and has preventative capacity against mastitis in dairy cows.

Evaluation of pollen tube growth ability in Petunia species having different style lengths.

Pollen tube growth is essential for the fertilization process in angiosperms. When pollen grains arrive on the stigma, they germinate, and the pollen tubes elongate through the styles of the pistils to deliver sperm cells into the ovules to produce the seeds. The relationship between the growth rate and style length remains unclear. In previous studies, we developed a liquid pollen germination medium for observing pollen tube growth. In this study, using this medium, we examined the pollen tube growth ability in Petunia axillaris subsp. axillaris, P. axillaris subsp. parodii, P. integrifolia, and P. occidentalis, which have different style lengths. Petunia occidentalis had the longest pollen tubes after 6 h of culture but had a relatively shorter style. Conversely, the pollination experiments revealed that P. axillaris subsp. parodii, which had the longest style, produced the longest pollen tubes in vivo. The results revealed no clear relationship between the style lengths and the growth rate of pollen tubes in vitro. Interspecific pollinations indicated that the styles affected pollen tube growth. We concluded that, in vitro, the pollen tubes grow without being affected by the styles, whereas, in vivo, the styles significantly affected pollen tube growth. Furthermore, interspecific pollination experiments implied that the pollen tube growth tended to be suppressed in the styles of self-incompatibility species. Finally, we discussed the pollen tube growth ability in relation to style lengths.

Roles of mTOR in the Regulation of Pancreatic ß-Cell Mass and Insulin Secretion.

Pancreatic ß-cells are the only type of cells that can control glycemic levels via insulin secretion. Thus, to explore the mechanisms underlying pancreatic ß-cell failure, many reports have clarified the roles of important molecules, such as the mechanistic target of rapamycin (mTOR), which is a central regulator of metabolic and nutrient cues. Studies have uncovered the roles of mTOR in the function of ß-cells and the progression of diabetes, and they suggest that mTOR has both positive and negative effects on pancreatic ß-cells in the development of diabetes.

The necessity of routine screening for deep vein thrombosis before surgery.

Background: Pulmonary embolism (PE) from deep venous thrombosis (DVT) can be a fatal postoperative complication. Preventive measures for venous thromboembolism (VTE) was evaluated in this hospital. Materials and methods: Preoperative DVT screening following surgery under general anesthesia in 2009-2016 was examined, and then, 217 patients diagnosed with DVT by preoperative leg-ultrasound (US) between 2014 and 2016 were retrospectively analyzed. Results: There were 24,826 operations under general anesthesia in the study period. Preoperative leg-US was performed in 5345 (21.5%) patients, and 648 (12.1% of patients, 2.6% of total operations) were diagnosed with DVT. In 2014-2016, 217 patients, which is 11.7% of patients undergoing leg-US, were diagnosed with DVT. DVT was found in the proximal veins (upper popliteal vein) in 86 (39.6%) patients. A total of 143 (62%) patients were considered to have organized thrombi, no patient developed pulmonary embolism, and 133 (58%) patients were discharged without follow-up examination for DVT. Ninety-six patients were evaluated for changes on leg-US, with no difference in the results with and without anticoagulant use. On multivariate logistic regression analysis, anticoagulants appeared effective for non-organized thrombi, higher D-dimer levels (≥10 µg/mL), or orthopedic surgery. Conclusion: Preoperative screening for DVT did not appear useful, and treatment of asymptomatic DVT was not always necessary.

Effects of Trehalose Solutions at Different Concentrations on High-Intensity Intermittent Exercise Performance.

Trehalose solution ingested during exercise induces gradual increases in blood glucose levels and the insulin response compared with glucose solution. Trehalose solution aids in the maintenance of performance in the later stages of prolonged exercise. The purpose of this study was to identify the lowest concentration at which the properties of trehalose could be exploited. Groups of 12 healthy men (21.3 ± 1.3 years) and 10 healthy men (21.1 ± 0.7 years) with recreational training were included in experiments 1 and 2, respectively. Both experiments followed the same protocol. After fasting for 12 h, the participants performed a 60 min constant-load exercise at 40% V˙O2 peak using a bicycle ergometer and ingested 500 mL of a trial drink (experiment 1: water, 8% glucose, and 6 or 8% trehalose; experiment 2: 4 or 6% trehalose). They performed four sets of the Wingate test combined with a 30 min constant-load exercise at 40% V˙O2 peak. The experiment was conducted using a randomized cross-over design; trial drink experiments were conducted over intervals of 7 to 12 days. The exercise performance was evaluated based on mean power in the Wingate test. Blood was collected from the fingertip at 12 points during each experiment to measure blood glucose levels. During the high-intensity 5 h intermittent exercise, no differences were found between the groups in exercise performance in the later stages with concentrations of 8, 6, and 4% trehalose solution. The results suggest that trehalose could be useful for making a new type of mixed carbohydrate solution. Further studies to determine the trehalose response of individual athletes during endurance exercise are needed.

Eating Perception, Nutrition Knowledge and Body Image among Para-Athletes: Practical Challenges in Nutritional Support.

Limited information exists on dietary practices in para-athletes. The aim of this study was to clarify the actual situation of para-athletes' dietary practice and to sort out the factors (i.e., eating perception, nutrition knowledge, and body image), that may hinder their dietary practices, and explored the practical challenges in nutritional support and improving nutrition knowledge for para-athletes. Thirty-two Japanese para-athletes (22 men) and 45 collegiate student athletes without disabilities (27 men) participated in the online survey. The questionnaire included demographic characteristics, eating perception, dietary practices, and nutrition knowledge. The Japanese version of the body appreciation scale was used to determine their body image. Para-athletes who answered that they knew their ideal amount and way of eating showed significantly higher body image scores (r = 0.604, p < 0.001). However, mean score for nutrition knowledge of para-athletes were significantly lower than collegiate student athletes (19.4 ± 6.8 vs. 24.2 ± 6.1 points, p = 0.001). Both groups did not identify a dietitian as the source of nutrition information or receiving their nutrition advice. The results indicate para-athletes have unique eating perceptions and inadequate nutrition knowledge. Future interventions are needed to examine nutritional supports and education in relation to the role of dietitians.