Background. Overexpression of decoy receptor 3 (DcR3) have been reported in various classes of malignancies. However, its expression and clinicopathological contribution in gliomas has not been fully elucidated. Objective. To explore the expression and clinical significance of DcR3 protein in relation to tumor cell differentiation and proliferation in glioma cell lines and tissues. Methods. One hundred and twenty-five samples of glioma patients and 18 cases of normal brain tissues were recruited. The expression of DcR3 protein was detected using immunohistochemistry. Tumor differentiation was assessed by histologic characters and the status of glial fibrillary acidic protein (GFAP). Tumor cell labeling indexes (LIs) of Ki-67 and PCNA were also obtained. The relationship between the DcR3 level and clinicopathological features was investigated, including tumor differentiation, LIs, and survival. Meanwhile, the expression of DcR3 protein was also measured in the supernatants of 8 glioma cell lines and glioma cells freshly prepared from 8 human glioblastoma specimens by using western blot. Results. The level of DcR3 protein in gliomas was significantly higher than that in normal brain tissues (P < 0.01). DcR3 expression showed positive correlations with tumor pathological grade  and negative with GFAP expression . Furthermore, there were positive correlations between DcR3 expression and Ki-67, PCNA LIs . The survival in the DcR3 negative group was 50 ± 1.79 months, longer than that of the DcR3 positive group (48.36 ± 2.90), however, without significance (P = 0.149). Different levels of DcR3 could also be detected in the culturing supernatants of all the 8 glioma cell lines and glioma cells freshly obtained from 8 human glioblastoma specimens. Conclusions. The overexpression of DcR3 might play a crucial role in the tumorigenesis, differentiation, and proliferation of glioma. DcR3 gene amplification, level of DcR3 mRNA, and protein in 46 astrocytic brain tumors by quantitative genomic PCR, quantitative reverse transcription-PCR, and immunohistochemistry, respectively. The study included 6 cases of stage II, 16 cases of stage III, and 24 cases of stage IV of gliomas. No normal brain tissues were covered in the aforementioned 2 studies. Hwang et al. [The decoy receptor 3 (DcR3), locus on 20q13, is a member of the tumor necrosis factor receptor (TNFR) superfamily . Previoug et al.  comparedn = 49). Grade III included 12 cases of anaplastic astrocytomas and 16 cases of anaplastic oligodendrogliomas, while all the 48 cases of grade IV were glioblastomas. Thus, the high-grade group comprised 76 subjects. Seventy-six patients were followed up to 5∼58.2 months. Till the endpoint of follow-up, 10 candidates were dead and overall survival (OS) was calculated. The 18 normal tissues were obtained from decompressive resections of traumatic brain injuries. The age of the normal controls ranged from 15 to 57 years, with a mean age of 34 years. All cases were initial tumorectomies without treatment and randomly selected in the First Affiliated Hospital of Guangxi Medical University, China, between July 2003 and October 2007. The study protocol was approved by the Ethical Committee of the First Affiliated Hospital of Guangxi Medical University. Written informed consent was obtained from the patients and clinicians for the usage of the samples for research. All samples were reviewed and diagnosed by two independent pathologists.This retrospective study included 125 cases of FFPE gliomas and 18 normal brain tissues. The age of the glioma patients ranged from 19 to 78 years, with a mean age of 54 years. Clinicopathological information was provided from medical records. In the current study, 10 cases were pilocytic astrocytomas (WHO grade I) and 39 cases were grade II, including 16 cases of fibrillary astrocytomas, 14 cases of serous astrocytoma, and 9 cases of oligodendrogliomas. These cases were classified into low-grade group for differentiation  were purchased from the American Type Culture Collection . All cell lines were cultured in Dulbecco's modified essential medium  and supplemented with 10% heat-inactivated fetal bovine serum , 2 mM glutamine, and gentamicin at 37°C in a humidified incubator with 5% COμm cell strainers to eliminate cell debris. After centrifugation and lysis of erythrocytes by washing with hypotonic water, the glioma cells were washed and resuspended in full medium of DMEM. Conditioned medium was harvested after no more than 5 passages.Preparation of primary glioma cell cultures was performed as reported by Roth et al. . Briefly6) were cultured in serum-free DMEM for 24 h. The supernatants were harvested and subsequently concentrated by centrifugal filter devices. The supernatants of freshly isolated ex vivo glioma cells were prepared accordingly [μg of protein was subjected to SDS-PAGE (12 SDS-acrylamide gel) with a loading buffer containing 80 mM Tris-HCl (ph 6.8), 5% SDS, 10% glycerol, 5 mM EDTA (ph 8), 5% 2-Mercapto Ethanol, 0.2% Bromophenol blue, and 1 mM phenylmethylsulfonyl fluoride. The separated proteins were transferred to PVDF membranes (Bio-Rad) for 2 hrs at 100 mA. The membrane was incubated with a DcR3 mouse monoclonal antibody  or a β-actin antibody . Primary antibodies were detected with an HRP-conjugated secondary antibody  and finally the membranes were subjected to chemiluminescence detection assay. Cell lysis sample from known DcR3 positive hepatocellular carcinoma cell line HepG2 was used as a positive control for western blot.Glioma cells  for Ki-67 and PCNA LIs. One-Way Analysis of Variance (ANOVA) test and Student's paired t-test were used to analyze significance between groups. The Least Significant Difference (LSD) method of multiple comparisons between 2 groups was applied when the probability for ANOVA was statistically significant. Kaplan-Meier and log-rank test were performed for the survival analysis. Statistical significance was determined at a P < 0.05 level.SPSS19.0  was used for statistical analysis. Mann-Whitney P < 0.01, r = 0.621, P < 0.01, P < 0.01). The strongly positive cases with (+++) were only observed in high-grade gliomas. Additionally, a negative relationship was noted between the DcR3 level and GFAP expression , which further indicated the correlation between DcR3 expression and tumor cell differentiation. No relative relationship was found between DcR3 and age or gender .DcR3 expression was found in 79.2% (99/125) of glioma patients versus 11.1% of normal controls . As expected, the negative correlations between the LIs and GFAP were found . Ki-67 LI was positively correlated to PCNA LI , however, with a slightly higher value . Both LIs of Ki-67 and PCNA were lower in the DcR3 negative group than that in the DcR3 positive group (P < 0.01), and they increased with the upregulation of DcR3 expression .The LIs of Ki-67 and PCNA were significantly lower in the normal brain tissues as compared to the gliomas. Both LIs increased with the growth of pathology grade . There wpression . Thus, tn = 19) was 50.00 ± 1.79 months, slightly higher than that of the DcR3 positive group . However, this difference did not reach the statistical value . When looked into the subgroups of DcR3 positive cases, we found that the shortest survival was of 26.29 ± 3.74 months for the strongly positive (+++) group, which was significantly different compared to the DcR3 negative group. However, the P value was 0.107 for the comparison of intergroup  with 19 cases being strongly positive expression. There was a positive correlation between DcR3 expression and the tumor grade , in agreement with other malignancies [r = −0.489, P < 0.01), which further indicated the close relationship between DcR3 level and the tumor differentiation.Next, we studied the correlation between DcR3 expression and glioma differentiation, which was evaluated by histological features and GFAP staining. Two research groups have reported the relationship between DcR3 level and pathological grade in glioma. DcR3 was found positive in 15 among 18 gliomas of grade IV (83%) by Roth et al. , while nin vitro and in vivo studies are needed to investigate the molecular mechanisms of DcR3 influencing the malignant phenotypes of gliomas.PCNA was originally identified as an antigen that is expressed in the nuclei of cells during the DNA synthesis phase of the cell cycle. The Ki-67 antigen can be exclusively detected within the cell nucleus during interphase, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. Ki-67 protein is present during all active phases of the cell cycle  but is absent from resting cells (G0). Both of PCNA and Ki-67 are excellent biomarkers to determine the growth fraction of a given cell population. The fraction of positive tumor cells (the labeling index) is often correlated with the clinical course of cancer. In the current study, we found that LIs of Ki-67 and PCNA rose with the increase of pathology grade. Meanwhile, positive correlations were found between DcR3 expression and Ki-67, PCNA Lis; that is, in the higher actively proliferated gliomas, the positive expression of DcR3 showed stronger expression, which indicates that DcR3 is closely related to the proliferation of glioma cells. Further We also attempted to explore the impact of DcR3 on patient survival. Among the 76 cases with follow-up, we found that the survival of the DcR3 negative group was slightly longer than that of the DcR3 positive group (50 ± 1.79 versus 48.36 ± 2.90 months). However, this difference did not reach the significant criterion. The possibility for DcR3 to act as prognostic biomarker for gliomas needed further investigation. For instance, more evidence should be obtained with a cohort with a bigger number of subjects investigated.in vitro and in vivo studies are planned to explore the contribution and mechanism of DcR3 in the malignant phenotype of human glioma cells.Together with previous studies, the current findings further confirm the role of DcR3 as an oncogene during the tumorigenesis and deterioration of human glioma. DcR3 expression in FFPE samples might be a prognostic biomarker for the differentiation and proliferative status of glioma cells. However, a larger cohort is warranted to verify the evidence. Further


This longitudinal research study aimed to develop a pregraduation clinical training program for nursing students before graduation and evaluate its effect on students' self-perceived clinical competence, clinical stress, and intention to leave current job. A sample of 198 students returned the questionnaires before and after the program. They were followed up at 3, 6, and 12 months after graduation. Results showed that posttest clinical competence was significantly higher than pretest competence, positively related to clinical competence at 3 and 12 months, and negatively related to clinical stress at 3 months. The clinical competence at 3 months was positively related to clinical competence at 6 and 12 months, and clinical competence at 6 months was related to intention to leave at 12 months. Intention to leave at 6 months was positively related to intention to leave at 3 and 12 months. Clinical stress at 3 months was positively related to clinical stress at 6 and 12 months, but not related to intention to leave at any time points. The training program improved students' clinical competence. The stressful time that was correlated with new graduate nurses' intention to leave their job was between the sixth and twelfth months after employment. Nurse shortage has become a worldwide issue. Using the United States as an example, the shortage of hospital registered nurses will reach 340,000 nurses by the year 2020 . Nurse tAlthough recruiting new graduate nurses was identified as important, according to Benner's Novice to Expert Theory , new graKramer  coined tThis study focused on Kramer's honeymoon and shock phases because these are regarded as the most difficult phases for new graduate nurses . The desA substantial body of research findings indicates that the transition of new graduate nurses from an academic program to a clinical setting is a stressful time period . New graAccording to Casey et al.  new gradResearchers  pointed Studies have focused on the relationship between clinical stress and intention to leave among new graduate nurses, but little attention has been paid to the relationships between clinical competence, clinical stress, and intention to leave all together in a longitudinal study. Clearly, the new employment orientation period encompasses substantial learning and is a stressful time for new graduate nurses. Therefore, the purpose of this study was to develop a pregraduation clinical training program for senior nursing students in their last semester before graduation and evaluate the effects of this program empirically and longitudinally on students' self-perceived clinical competence, clinical stress, and intention to leave their current job. The conceptual framework guiding this study is illustrated in The pregraduation clinical training program was designed to bridge the gap between student and professional nurse roles and was implemented for the first time at our school when this study started. This program was a three-credit hour course required for graduation. Participants were students in a two-year baccalaureate degree nursing program (RN-to-BSN). They had previously completed a five-year diploma nursing program and were licensed to practice nursing. However, these students directly entered the RN-to-BSN program as full-time students without any nursing work experience. The aim of the program was to provide clinical placements at hospitals where students wanted to get jobs following graduation. The training program thus served to facilitate enhanced competence development and also helped nursing students become familiar with their future work place while they were still students. This familiarity might reduce new graduates' experience of reality shock and prevent turnover, avoiding costs for hospitals. Therefore, the school faculty had meetings with students first to determine their preferred hospitals for practice. However, based on the school's regulation that all hospitals where students had clinical practicum must pass the evaluation by the Department of Health in Taiwan, not all students' preferred hospitals could be selected. We then visited each of those students' preferred hospitals and explained the purpose and content of the pregraduation clinical training program with hospital administrators.The course required students to practice as a registered nurse under the supervision of a hospital registered nurse preceptor for a total of 240 hours within six weeks. Each student was assigned with a preceptor during the program and scheduled to work the same schedule with their preceptor, which had to include at least two different kinds of shifts. To qualify as preceptors, nurses at the clinical facilities had to have passed a preceptor training program held by their facility. During the pregraduation clinical training program, the school faculty served as external mentors, facilitating student nurses' clinical learning and acting as a coordinator between the school and the clinical facilities. Faculty conducted four-hour discussion meetings biweekly with all the students in the program. These discussions provided opportunities for students to share experiences with each other and served as a channel for communication between students, faculty, and clinical facilities.The study was a longitudinal design. All students received the designed training program. Data was collected before implementation of the training program (pretest), after the conclusion of the training program (posttest), and at three, six and twelve months after students graduated and began their first year of employment.Two hundred six pregraduates students enrolled in the final semester of the two-year RN-to-BSN program in the investigators' school were contacted and invited to participate in the study and 198 students completed the pretest and posttest questionnaires, and 111, 101, and 75 new graduate nurses returned questionnaires in the third, sixth, and twelfth months after they graduated and began their first job. The study response rate was 96.12% in the pretest and posttest and was 53.88%, 49.03%, and 36.41% in the third, sixth, and twelfth months after graduation. The participants completed all questionnaires at places they felt comfortable at.The data collection started after a local research ethics committee in Taiwan approved the study protocol. Following the approval, we held a meeting with the students to explain the program and the research study, including the study purpose, procedures, participants' rights, and confidentiality. This information was also included in the cover letter that was sent to the participants along with a set of questionnaires and a self-addressed-and-stamped envelope before each data collection time point. Although the participants were not required to sign and return a consent form, finishing and returning the questionnaires indicated participants' agreement to participate in this study. For participants' convenience, they could choose to fill out the questionnaires either in a paper-and-pencil form or in an electronic form. If the participants chose to use the paper-and-pencil form, they had to mail back the completed questionnaires using the provided envelope. If the participants chose to use email, they had to reply to the invitation email sent by the research team and attach the completed questionnaires to their emails. Emails that could identify the participants were deleted after the electronic completed questionnaire was saved in a secured computer that was protected with passwords known only to the primary investigator.The Clinical Competence Questionnaire (CCQ). The CCQ is an instrument developed by Liou and Cheng [nd Cheng  to evalund Cheng . CronbacThe Clinical Stress Scale (CSS). The CSS is an instrument developed by the investigators based on the literature reviews to evaluate new graduate nurses' level of clinical stress. The CSS is a 46-item 5-point Likert-type scale (ranges from 0 to 4). Sample questions are “I have knowledge and skills to implement nursing care” and “I can identify and apply related work resources.” A higher score indicates a higher level of stress. Validity of the scale was confirmed by exploratory factor analysis that showed 76.28% of the variation in clinical stress could be explained. Cronbach's alpha of the CSS was .95 in this study.The Anticipated Turnover Scale (ATS).   The new graduates' intention to leave their current job was measured by the Anticipated Turnover Scale (ATS), which was developed by Hinshaw et al. [w et al. . The purw et al. . In thisP values less than .05 were considered statistically significant. All students in this study received the designed pregraduation clinical training program and therefore the program was not treated as an independent variable. Participants' demographic information and levels of major measured variables were analyzed by descriptive statistics. Normality of the measured variables was tested using the Shapiro-Wilk test. Results showed that clinical competence at all measuring time points and intention to leave in the third and twelfth months were not normally distributed. Therefore, Spearman's correlation was used to examine relationships between measured variables that were not normally distributed whereas Pearson correlation was used for normally distributed variables. The Generalized Estimating Equations analysis introduced by Liang and Zeger [All data were entered and analyzed using SPSS version 17.0. All tests were 2 sided and nd Zeger  was usednd Zeger .All participants were females with an average age of 23 years. Upon the completion of the pregraduation clinical training program, only 3.9% of students felt dissatisfied with their performance during the training program. Almost all students were satisfied with the school's arrangement of the program (98.5%), 96.5% felt that the hours of the training program were appropriate, and 99% were satisfied with the preceptorship during the practice.As shown in Participants rated their clinical competence as high and their clinical stress and intention to leave their job as medium low across all data collection time points . The GenParticipants experienced median high level of clinical stress within one year of employment but did not have high intention to leave their current job . Comparer = .35,  P < .001) and 12-month competence  but not 6-month competence. The 3-month competence score was positively correlated with 6-month competence  and 12-month competence , and 6-month competence was positively correlated with 12-month-competence . The post-competence was negatively correlated with clinical stress only at three months , but was not correlated with intention to leave at any time point. The 3-month-competence was negatively correlated with clinical stress at three months  but was not correlated with intention to leave at any time point. The 6-month competence was correlated with intention to leave at twelve months . The clinical stress at three months was positively correlated with clinical stress at six months  and clinical stress at twelve months  but was not related to intention to leave at any time point. The clinical stress at six months was positively correlated with clinical stress at twelve months  and intention to leave at six months . The intention to leave at three, six, and twelve months was positively intercorrelated . The intention to leave at three months was not related to clinical competence and clinical stress at any time point.n = 41, M = 195.80, SD = 25.25), clinical stress , and intention to leave  in the third month of the participants who remained in the hospital where they completed the pregraduation clinical training program and the scores of those who did not remain  did not show difference .Scores of clinical competence (Study results reveal that, in general, the pregraduation clinical training program was effective in increasing students' clinical competence. This result is consistent with the other research findings that developing special programs to bridge the gap between academic education and clinical practice can improve new graduate nurses' clinical competence , 28, 29.This study also found that students' clinical competence after the pregraduation clinical training program and in the third month after employment was negatively correlated with clinical stress in the third month after employment. During this period, new nurses may have difficulties in adjusting to their new roles and may experience an increase in stress associated with job expectations and lower clinical competence. In addition, new graduate nurses experienced median high level of stress and the stress remained about the same level within one year of employment. This result is similar to results of previous studies suggesting that the time frame between three and six months was the most stressful time for new graduate nurses and that caused new graduate nurses to have strong intention to leave their current job , 30. HowThe nonsignificant relationship between clinical stress, competence, and intention to leave could possibly be explained by other reasons. The new graduate nurses had worked for only three months and might have rationalized their lower clinical competence and higher stress as unfamiliarity with the work environment. They may have considered that it was too soon to feel completely competent and thus were not feeling at that time any intention to leave their current job. These results also confirmed the Reality Shock Theory that new nurses in the phase one honeymoon stage focused more on the excitement and euphoria of having a real nursing job and about getting paid for the work.The results of this study presented evidence that new graduate nurses benefited from the structured pregraduation clinical training program, including an improvement in clinical competence. However, generalisability of the study's findings is limited because only students studying in the two-year RN-to-BSN nursing program in one university were included in the sample. To increase the generalisability of the results, it is recommended that future investigations include nursing students in other nursing programs such as the traditional four-year Bachelor's program or five-year diploma program. This study did not have a comparison group who completed a regular clinical practicum, because we wanted to let all students equally have the chance to participate in the program. Although this study found that new graduate nurses who participated in the pregraduation clinical training program had significantly higher posttest clinical competence score compared to pretest, the strength of the conclusion drawn—that the program results in significant increase in clinical competence—is limited by the lack of a comparison group in this study. Therefore, it is recommended that future research includes a comparison group that does not participate in the pregraduation clinical training program to increase the power of the conclusion. Qualitative interviews of the unit managers may also provide valuable insight, and it is suggested that future studies include these interviews to support the quantitative findings by furnishing an in-depth and alternative perspective of the situation.This study provides meaningful information to nurse educators and healthcare administrators. The results revealed that the pregraduation clinical training program started nurse students' learning about how to be a professional nurse before they left school, was effective in promoting students' clinical competence, and was conducive to nurse students' adaptation to their clinical jobs following graduation. New graduate nurses' clinical competence remained higher after the program than before the program. Although new graduate nurses' clinical competence dropped insignificantly in the third month, it increased in the sixth and twelfth months. There are two implications for this result. First, the pregraduation clinical training program might play an important role to subdue a certain degree of reality shock during these months and help new graduate nurses regain higher levels of competence quickly. Second, the period from postgraduation to the sixth month is a critical time for new graduate nurses to reinforce clinical competence. Hospitals that can provide a supportive environment and training that meets new graduate nurses' needs during this period can help nurses establish higher levels of competence and decrease the subsequent clinical pressure. Finally, this study revealed that the most stressful time was the first three months, but the stressful time that was correlated with new graduate nurses' intention to leave their job was between the sixth and twelfth months after employment. This finding has significant implications for healthcare administrators. The golden time for hospitals to promote new graduate nurses retention must start at the beginning of employment, or this high level of stress may be accumulated over time and, finally, causes nurses to leave their jobs. More importantly, hospital administrators should not view new graduate nurses as an immediate solution to the nurse shortage. Instead, a long-term strategy should be applied, aiming at cultivating the professional development of new graduate nurses over at least twelve months. This may be the golden rule for successful retention of new graduate nurses.


Polygonum minus were fractionated using an eluting solvent to evaluate the pharmacological mechanisms underlying the anti-ulcerogenic activity of P. minus. Different P. minus fractions were obtained and evaluated for their ulcer preventing capabilities using the ethanol induction method. In this study, Sprague Dawley rats weighing 150–200 g were used. Different parameters were estimated to identify the active fraction underlying the mechanism of the gastroprotective action of P. minus: the gastric mucus barrier, as well as superoxide dismutase, total hexosamine, and prostaglandin synthesis. Amongst the five fractions from the ethanolic extract of P. minus, the ethyl acetate:methanol 1:1 v/v fraction (F2) significantly (p < 0.005) exhibited better inhibition of ulcer lesions in a dose-dependent manner. In addition, rats pre-treated with F2 showed a significant elevation in superoxide dismutase (SOD), hexosamine and PGE2 levels in the stomach wall mucosa in a dose-dependent matter. Based on these results, the ethyl acetate:methanol 1:1 v/v fraction was considered to be the best fraction for mucous protection in the ethanol induction model. The mechanisms underlying this protection were attributed to the synthesis of antioxidants and PGE2.The leaves of  Helicobacter pylori, smoking, anti-inflammatory drugs, and pepsin production [Polygonum minus is a plant belonging to the family Polygonaceae and is locally called kesum in Malay [P. minus possesses high free radical scavenging activity and reducing power using different extracting solvents [P. minus in order to identify its phytochemical constituents because it is highly aromatic and rich in essential oils [in vitro [in vivo [P. minus has been used to treat digestive disorders. Pharmacologically, P. minus has been reported to possess antimicrobial [Peptic ulcer is a common digestive disease and is considered to be a major cause of morbidity and mortality . Scientioduction –4. Most in Malay . This plsolvents ,7. Reseaial oils . In addi[in vivo , it can icrobial , antiviricrobial .P. minus. Following the traditional use of P. minus to treat digestive disorders, this study was conducted to evaluate for the first time the pharmacological mechanisms underlying the anti-ulcerogenic activities of the fractions obtained from an ethanolic extract of P. minus using different analytical grade solvents.In the current study, the ethanol induction model was used as it has been reported as a rapid and convenient model of gastric ulcer induction in experimental animals. As well, ethanol is considered to be a necrotizing factor that contributes to the formation of lesions in the gastric mucosa . The mecP. minus reduced ulcer area significantly (p < 0.05) when compared with the CMC (carboxymethyl cellulose) negative control group presented in All the fractions from the ethanolic extract of P. minus, using different analytical HPLC grade solvents, to evaluate the mechanism of gastroprotection mediated by this plant. Peptic ulcer is postulated to be a multifactor disease, but each factor acts via a particular mechanism to contribute to the aetiology of the mucus lesion [The present study focused on fractions of s lesion . The mecs lesion , which as lesion . In conts lesion ,18.The current medical treatment of peptic ulcer is in general based on the enhancing of mucosal defense against necrotizing effects such as alcohol as well as none steroidal anti-inflammatory drugs (NSAIDs). However, one of the major problems in gastro-duodenal ulcer treatment is that despite the healing rate of 80–100% after 4–8 weeks of therapy with H2 antagonists and proton pump inhibitors, the rate of ulcer recurrence within 1 year after stopping management is between 40% and 80% . BesidesSeveral beneficial plant extract are used to enhance the mucosal defense mechanism, shift in proton pump, stabilizing surface epithelial cells or interfering with prostaglandin synthesis . MoreoveP. minus promoted better inhibition of lesions in the gastric mucosa compared to the other fractions  between these groups and the omeprazole positive control group.Gastric mucus wall production after pre-treatment with all the fractions was increased in a dose-dependent matter, as shown in p < 0.05) in all groups compared to the CMC negative control group. The F2 pre-treated group exhibited no significant difference with the PGE2 synthesis (123.9 pg/mL ± 5.52) in comparison with the omeprazole positive control group  different compare to CMC negative control group. The rats pretreated with F2 (82.95 μg/g ± 3.45) showed highest level of hexosamin and there is no significant difference (p < 0.05) with omeprazol positive control group (81.60 μg/g ± 5.41)  .et al.[The ulcer prevention activity of phenolic compounds and their derivatives, notably demonstrated in various gastric induction models, have been postulated to occur by mechanisms that include the stimulation of prostaglandin synthesis, the elevation of mucus production and the reduction of gastric pH . Accordiet al..p < 0.05) promoted scavenging activity against ethanol-induced ulcers. In contrast, F1 at 50 mg/kg, as well as F3 and F4 at both treated doses showed significantly different results (p < 0.05) compared to the omeprazole treated group.Enzyme scavenging activity, particularly via SOD, as presented in in vitro antioxidant activity of P. minus [in vivo enzymatic scavenger such as SOD. This enzyme is considered as the first line of defence against reactive oxygen species [In addition, we assessed the antioxidant enzyme level as a gastroprotection mechanism. As mentioned previously, oxidative stress is one of the main causes of gastric ulcers induced by ethanol. Hence, antioxidants are hypothesized to be one of the prevention factors against ethanol-induced ulcers. Previous work showed the powerful P. minus ,32. Ther species . The resP. minus fraction (F2). It was shown that F2 was the fraction with highest amount of phenolic compounds such as gallic acid, rutin, coumaric acid and quercetin as presented in The phytochemical analysis were performed using HPLC and UPLC-ESI-MS/MS, High performance liquid chromatography analysis determined the phenolic compounds contained in the ethanolic extract of P. minus were obtained from Ethno Resources Sdn Bhd, Selangor Malaysia, The plant was identified, and voucher specimen was kept in our laboratory for future references. Air-dried powdered leaves of P. minus were extracted by drenching in 95% ethanol  at room temperature for 3 days. Afterwards, the solvent was filtered, followed by concentration under reduced pressure until excess solvent was removed. The resulting yield was 5.8 g/100 g. Next, 1.5 g of the crude extract was chromatographed over silica gel  using FLEX™ columns. Different fractions were obtained from the stepwise elution of different ratios of hexane, ethyl acetate, methanol, acetonitrile, and distilled water. Depending on the TLC profile, similar fractions were combined. Ultimately, five fractions were obtained from P. minus leaf extract. The first fraction, F1, was eluted with hexane:ethyl acetate 1:1 v/v to give 0.267 g. The second fraction, F2, was obtained using ethyl acetate:methanol 1:1 v/v and gave 0.651 g. The third fraction, F3, was eluted with methanol:acetonitrile 1:1 v/v and gave 0.241 g. The fourth fraction, F4, was eluted with acetonitrile:distilled water 1:1 v/v yielding 0.20 g1:1 v/v. Finally, the fifth fraction, F5, was eluted with distilled water producing 0.12 g1:1 v/v. The gastroprotective ability of all of the fractions was evaluated in order to identify the most active fraction.Dried leaves of et al. [P. minus: F1, F2, F3, F4, and F5, respectively. After one hour, all rats were administered 95% ethanol 5 mL/kg and after an additional 60 min, the rats were sacrificed by an overdose of ketamine 100 mg/mL. The stomach was rapidly removed for gross and histological evaluation and biochemical testing.Following the method described by Garg, et al.  with min2) as described by Njar and Mahmood, et al. [Macroscopically, the lesions appeared as hemorrhage bands along the long axes of the stomach. The lesion of each stomach was calculated under a dissecting microscope (1.8×) using a hemocytometer (big square: length × width = 10 mm × 10 mm = ulcer area). The sum of the areas of all lesions for each stomach was implemented in the calculation of the ulcer area (UA) wherein the sum of small squares × 4 × 1.8 = UA  solution for histological examination. The fixed stomachs were processed, sectioned every 5 μm and stained with H & E.et al. [Gastric wall mucus was determined according to the procedure of Corne and Al-Qarawi, et al. ,37. The The stomachs were homogenized using ice cold Tris-HCl pH 8.2 (1 g in 10 mL) on ice. The homogenate tissues were centrifuged at 4500 g for 15 min at 4 °C, and then stored at −80 °C until they were used. In this study, the homogenate was analyzed in order to estimate superoxide dismutase, hexosamine and prostaglandin synthesis.Prostaglandin E2 (PGE2) levels produced in the gastric mucosa were measured following the procedure described in Cayman’s Protein Determination Kit.The concentration of hexosamine in stomach tissue was analysed according to the reported method of  with minStomach superoxide dismutase activity was measured at 460 nm using a plate reader following the method reported in Cayman’s Superoxide Dismutase Assay Kit.2O + 0.1% TFA) and solvent B (methanol + 0.1% TFA), at a unique flow rate of 1 mL/min. The gradient elution program was as follows: 0 min 90% A:10% B; 5 min 55% A:45% B; 20 min 50% A:50% B; 21 min 40% A:60% B; 22 min 90% A:10% B. The sample injection volume was 20 μL followed by equilibration run of 15 min and monitored with an SPD-20 A (UV) at 280 nm.Phytochemical analysis was conducted to identify phenolic compounds by using the high performance liquid chromatography (HPLC) method, using an LC-20AD apparatus  . The expet al. [The phenolic profiles were further confirmed by ultra-performance liquid chromatography-electrospray tandem mass spectrometry. This method is described in detail by Chua, et al. . To perfp < 0.05) using GraphPad PRISM®, version 5.0 [The data were analyzed using Statistical Package Social Science (SPSS), version 17.0 . One-waysion 5.0 .P. minus for its gastroprotective capability and it can therefore be formulated and recommended to be a therapeutic drug for gastric ulcer.The ethyl acetate:methanol fraction 1:1 (F2) is the best fraction for gastroprotective activity against oxidative stress caused by ethanol induction model. The efficacy of F2 is based on its high content of phenolic compounds, particularly gallic acid and coumaric acid, which contribute to antioxidant activity, mucus barrier as well as maintenance of PGE2 and SOD levels . This co


Aspergillus fumigatus clinical isolates. A full understanding of ergosterol biosynthetic control therefore appears to be essential for improvement of antifungal efficacy and to prevent antifungal resistance. An ergosterol biosynthesis pathway in A. fumigatus has been proposed with 14 sterol intermediates resulting in ergosterol and another secondary final compound C-24 ethyl sterol. Transcriptomic analysis of the A. fumigatus response to host-imposed stresses or antifungal agents is expanding our understanding of both sterol biosynthesis and the modes of action of antifungal drugs. Ultimately, the identification of new targets for novel drug design, or the study of combinatorial effects of targeting sterol biosynthesis together with other metabolic pathways, is warranted.Ergosterol, the major sterol of fungal membranes, is essential for developmental growth and the main target of antifungals that are currently used to treat fatal fungal infections. Emergence of resistance to existing antifungals is a current problem and several secondary resistance mechanisms have been described in  Aspergillus is azole drugs, such as voriconazole (VCZ) and itraconazole, and a new generation of azoles that includes posaconazole, isavuconazole, and albaconazole. Azoles revolutionized medical mycology due to their broad spectrum and reduced toxicity compared to AMB. Azoles block the ergosterol biosynthesis pathway via inhibition of 14-α sterol demethylase (Cyp51/Erg11), a key enzyme that removes the methyl group at position C-14 of precursor sterols. Inhibition of ergosterol synthesis at this biochemical level results in toxic sterol accumulation and cell death . Genetic and biochemical analysis of azole susceptible wild-type strains and a panel of clinical azole resistant strains identified a total of 14 sterols showing a similar sterol pattern composition, with ergosterol as the main sterol for all of them (Table 1). Each of the azole resistant strains had a different antifungal susceptibility pattern that correlated with different mutations in the cyp51A gene. The lack of differences between sterols highlights the fact that the resistance mechanism in those strains was target-dependent and not sterol-dependent, as will be discussed later. Instead, the sterol composition of single enzyme defective A. fumigatus mutants was rather different depending on the lacking enzyme (cyp51A and cyp51B) had less ergosterol than the parental strain and accumulated C-4, and C-14 methyl sterols; mainly eburicol . Accumulation of eburicol, together with a decrease in ergosterol, was much more prominent when the cyp51B gene was deleted compared to Δcyp51A strain. Downstream in the pathway, the synthesis of ergosterol involves the transformation of fecosterol, consisting of double-bond rearrangements in the steroid nucleus and in the side chain, isomerization of the double connection in the C-8 to the C-7 followed by the desaturation at C-5 and C-22, and the reduction of the C-24. The enzymatic sequence of these three last steps could differ between fungal taxa and growth conditions  and a vast accumulation of the Δ7 sterols . Although the sterol composition revealed differences according to the enzyme that was deleted, none of the single gene deletions, at 14-α sterol demethylase or C-5 sterol desaturase level, was lethal for A. fumigatus or influenced its virulence , together with the presence of two tandem copies of a 34-bp sequence in the promoter of the cyp51A gene. This latter type of resistance mechanism may have evolved in the environment through the exposure of the fungus to azole fungicides used in agriculture  or fungi static and more vulnerable to resistance (triazoles). Triazoles are inhibitors that target the ergosterol biosynthetic pathway by binding to the Cyp51 family of cytochrome P450s (14-α sterol demethylases) causing the depletion of ergosterol biosynthesis and the accumulation of lanosterol or eburicol . A. fumid cyp51B  that ared cyp51B . The letd cyp51B . Moreove studied . Also, t therapy . A. fumiutations , while riculture . In isoliculture , howeversertions . Furthersertions , howeveray Table .A. fumigatus susceptibility to AMB in vitro and clinical outcome is unclear. Secondary resistance to AMB is generally not observed, even in patients whose therapy has failed , a well-characterized naphthalene-degrading enzyme, suggesting that resistance could follow degradation of the naphthalene ring contained in terbinafine . Erg1 catalyzes the first oxygenation step in sterol biosynthesis and is suggested to be one of the rate-limiting enzymes in this pathway. Alterations in sistance , howeverbinafine . Also, asistance . Since tA. fumigatus initiates disease is particularly interesting. The transcriptome of A. fumigatus during early stages of infection in the neutropenic murine lung identified reduced expression of genes encoding ergosterol pathway functions  relative to laboratory cultures  showed that Erg24 and Erg25 functions were down-regulated for all of the time points, and in contrast Erg11 was up-regulated at 30 and 60 min of iron exposure . In S. cerevisiae the methylation is catalyzed by erg6 which converts zymosterol into fecosterol, and at least two methyltransferases are involved in consecutive methylation reactions leading to 24-ethyl sterols in higher plants (A. fumigatus, only one Erg6 has been identified that could transform lanosterol to eburicol and may also be involved in the production of C-24 ethyl sterols. The sterol profile analysis of A. fumigatus strains showed that the synthesis of 24-Ethylcholesta-5,7,22-trien-3β-ol is sensitive to pathway perturbations. In a Δcyp51B mutant a lack of ergosterol is compensated by an increase of 24-Ethylcholesta-5,7,22-trien-3β-ol, probably because the activity of the pathway becomes diverted toward the synthesis of alkyl sterols. However, when erg3B is suppressed, ergosterol levels are decreased together with 24-ethylcholesta-5,7,22-trien-3β-ol, confirming that this enzyme would also be involved in the desaturation of C-5,7 alkyl sterols.Collectively, the above observations suggest that ergosterol biosynthesis is prone to perturbation by environmental conditions, and in a manner dependent upon Erg6, Erg11, Erg24, Erg25, and Erg3 functions, thereby highlighting these gene functions as alternative or synergic inhibitors of the pathway. An important finding in r plants . In A. fA. fumigatus is able to synthesize ergosterol, even with suppression of several enzymes in the pathway. Biosynthetic control under different environmental circumstances is observed, demonstrating that Aspergillus has alternatives to overcome severe drawbacks. Therefore, the precise knowledge of this complex biosynthetic pathway could facilitate the future development of novel and more selective antifungal drugs in order to improve efficacy and to minimize drug resistance.Genome wide studies are providing us with a very useful platform from which to dissect the linkages between cellular sterol biosynthesis and other cell functions or metabolic pathways. Such new findings might be further explored for novel drug development or for possible combinatorial therapeutic strategies to fight invasive fungal diseases. Among them, genes encoding enzymes involved in cellular stress, cell wall synthesis, and transport have been found to be differentially expressed under antifungal exposure or underhost imposed stresses. To conclude, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Apart from direct protection and reduced productivity loss during epidemics, the main reason to immunize healthcare workers (HCWs) against influenza is to provide indirect protection of frail patients through reduced transmission in healthcare settings. Because the vaccine uptake among HCWs remains far below the health objectives, systematic programs are needed to take full advantage of such vaccination. In an earlier report, we showed a mean 9% increase of vaccine uptake among HCWs in nursing homes that implemented a systematic program compared with control homes, with higher rates in those homes that implemented more program elements. Here, we report in detail the process of the development of the implementation program to enable researchers and practitioners to develop intervention programs tailored to their setting.We applied the intervention mapping (IM) method to develop a theory- and evidence-based intervention program to change vaccination behaviour among HCWs in nursing homes.i.e., an outreach visit to all nursing homes, plenary information meetings, and the appointment of a program coordinator -- preferably a physician -- in each home. Finally, we planned program adoption, implementation, and evaluation.After a comprehensive needs assessment, we were able to specify proximal program objectives and selected methods and strategies for inducing behavioural change. By consensus, we decided on planning of three main program components, The IM methodology resulted in a systematic, comprehensive, and transparent procedure of program development. A potentially effective intervention program to change influenza vaccination behaviour among HCWs was developed, and its impact was assessed in a clustered randomised controlled trial. It has been well established that during influenza epidemics, the etiological fraction of culture or PCR-confirmed influenza virus in elderly patients is high -- between 55% and 67%  of 0.95). This model included two demographical determinants, nine behavioural determinants, and two organisational determinants in which odds ratios (OR) were reported as measures of associations . Furthermore, to support future implementation of the program -- without assistance by our study group -- a step-by-step script of the total program was developed. In addition, the plenary information meetings were held by specialised nurses of the local municipal health centre guided by a standardised protocol. In The Netherlands these municipal health centers have a supportive role in the prevention of infection prevention in general and specifically influenza. In this protocol, we also included a list of frequently asked questions and corresponding answers.To assure program adoption, implementation, and sustainability, stakeholders were approached to give feedback on and to support the program. Representatives of the Dutch association of nursing home physicians (Verenso) and the association of nurses and nursing assistants (V&VN) were approached to judge the different elements of the program. The V&VN is the sole society for all Dutch nurses and nursing assistants with 36,000 members . They were asked for feedback on usefulness of the program elements and if program elements could be improved. This feedback was used to fine-tune the program elements, mainly by adjustment of difficulty of the language used. We did not translate the written information into other languages because most nurses in nursing homes understand the Dutch language. Support to the program was given byVerenso, V&VN, and two other relevant healthcare management associations (Sting and ActiZ) and visualised with their logo on program materials  would then be asked to appoint preferably a physician as local program coordinator (component C). Next, all nursing homes would be visited in September to deliver the step-by-step script of the program, all required materials, and the background information on influenza vaccination of HCWs (component A). Following these visits, execution of all program activities would be planned for October and November prior to the actual immunization of HCWs. During this period, the plenary information meetings were to be held by a specialised nurse of the local municipal health centre (component B).Our evaluation plan included both an effect and a process evaluation. We planned to evaluate the effectiveness of the program on influenza vaccine uptake among HCWs in Dutch nursing homes by comparing uptake in a group of at least 12 nursing homes randomly allocated to receive the intervention program with the uptake in a similar number of control homes. For this, we planned to perform a clustered randomised controlled trial . In the This paper presents the process by which a theory- and evidence-based intervention was developed to improve influenza vaccination behaviour among HCWs. The IM method was used to systematically develop this intervention.A major strength of the developing process was the comprehensive and thorough needs assessment that clearly identified relevant determinants for influenza vaccine uptake among HCWs on both management and HCW level. Combining explorative and theory-based methods assured that determinants not anticipated beforehand were included in the needs assessment and helped broaden the scope of this needs assessment. Based on the results of the needs assessment, we were able to quantify the importance of the determinants. This provided an anchor for the specification of program objectives and the successful further development of the intervention.The selection of methods and strategies (step three of the IM method) is challenging, because specific objectives can ask for a variety of different interventions, and consensus is needed by the developers on the intervention with presumed highest impact . As yet,Our systematic, comprehensive, and transparent description of all the steps in the development of the program enables future users to assess and adapt the program where necessary or to replicate the steps described when developing a similar program for a different population. Applying the systematic, comprehensive, and transparent IM approach guided and facilitated our development process. It may seem elaborate and time consuming, but a review of health promotion intervention studies has shown that the quality of planning is important for the success of the intervention ,35. ReceThe authors declare that they have no competing interests.ILvdA conducted the questionnaire studies and wrote the paper. MEH supervised the design of the program and drafts of the paper. TJMV commented on the paper. JRD critically commented on the paper and revised earlier drafts. JJvD and EH contributed both equally to the design of the program, were involved in earlier drafts, supervised the project and final draft. All authors have read and approved the final manuscript.


The purpose of this study was to evaluate the effects of a circuit training program along with a maintenance program on muscular and cardiovascular endurance in children in a physical education setting. Seventy two children 10–12 years old from four different classes were randomly grouped into either an experimental group (n = 35) or a control group (n = 37) (two classes for each group). After an eight-week development program carried out twice a week and a four-week detraining period, the experimental group performed a four-week maintenance program once a week. The program included one circuit of eight stations of 15/45 to 35/25 seconds of work/rest performed twice. Abdominal muscular endurance (sit-ups in 30 seconds test), upper-limbs muscular endurance (bent arm hang test), and cardiovascular endurance (20-m endurance shuttle run test) were measured at the beginning and at the end of the development program, and at the end of the maintenance program. After the development program, muscular and cardiovascular endurance increased significantly in the experimental group (p < 0.05). The gains obtained remained after the maintenance program. The respective values did not change in the control group (p > 0.05). The results showed that the circuit training program was effective to increase and maintain both muscular and cardiovascular endurance among schoolchildren. This could help physical education teachers design programs that permit students to maintain fit muscular and cardiovascular endurance levels. Physical fitness is nowadays considered as one of the most important health markers in childhood . ConseqIt is known that planning long-term fitness programs is the best way to improve these components . NonethOther problem related to physical fitness is its expected decrease after a period of detraining. Several authors confirm that after 8 to 12 weeks of detraining children lose a significant part of the physical fitness gains obtained . A poss2) from four different PE classes of a public primary school participated in this study. For practical reasons and the nature of the present study  a cluster randomized controlled trial was used. General profiles were assigned randomly to form one of the study groups (two classes for each group): experimental group , or control group . EG and CG consisted of two gender balanced groups .Seventy two apparently healthy Spanish children  10–12 years old  and 27 children in the CG (73%) regularly participated (at least twice per week) in organized sports programs. Children and their legal guardians were fully informed about all the features of the study and were required to sign an informed consent form. The Ethical Committee of the University of Granada approved the study protocol.The participants were evaluated using the muscular and cardiovascular endurance tests included in the EUROFIT battery , validaThe tests were administered in an indoor sports center court with a non-slip floor, under the same environmental conditions, on the same day and at the same time for each student. A blind evaluation was carried out by two researchers following the standard protocol for each test. Each researcher assessed physical fitness with the same tests using identical equipment. Prior to the evaluation, the participants completed a standardized warm-up consisting of five minutes of running from low to moderate intensity. The order and a brief description of the test protocol are as follows:Sit-ups in 30 seconds test(SUP). This test was used to measure abdominal muscular endurance. The participants laid supine on the mat with their knees flexed at an angle of 90 degrees and their feet flat on the floor, stabilized by a researcher. The participant’s fingers were to be interlocked behind their head. On the command ‘Go’, the participants’ elbows had to contact the knees and return to the starting position as many times as possible in 30 s. Each participant was allowed to perform the test once. The total number of sit-ups performed in 30 s was recorded.Bent arm hang test(BAH). This test was used to measure upper-limbs muscular endurance. The participants had to maintain a bent arm position while hanging from a bar with hands in a pronated grip and at shoulder width. The participants’ chin had to be above the bar and held in this position as long as possible. The test ended when the participants’ eyes went below the bar. Each participant was allowed to perform the test once. The total time in seconds was retained.20-m endurance shuttle run test(ESR). This test was used to assess cardiovascular endurance. All students ran between two parallel lines put 20 m apart, in the rhythm marked by a recorded beep. A researcher ran alongside the children to help them keep the desired pace. The starting speed was 8.5 km/h; and it increased 0.5 km/h every minute. The test ended when the child stopped running due to fatigue or failed to reach the line before the next signal for two consecutive times. Each participant was allowed to perform the test once. The last completed lap (timed in seconds) was recorded.A circuit training program was applied to the EG during the PE classes under the supervision of a researcher. Firstly, the EG participants performed a development circuit training program twice a week on nonconsecutive days for eight weeks. They completed a total of 14 training sessions, since two classes coincided with festivals and could not be used. Then, after a period of detraining (four weeks) coinciding with Christmas holiday, the EG participants completed a maintenance circuit training program one session per week during four weeks. During the period of maintenance program, each session of maintenance was alternated with a normal class of PE according to the course planning designed by the teacher.Each session lasted 50 minutes and consisted of a five-minute warm up during which children had to play a racing game, 40-minute circuit training, and two series of a 15–30 second cool-down of static stretching, primarily for the hamstrings and lumbar region . All exDuring the work time the students should complete as many repetitions as possible in a controlled manner. As other studies show, the last repetition of each set represents the momentary muscular fatigue . In ordDuring the development and maintenance programs the EG executed the circuit training,while the CG participated in traditional games, basketball and volleyball activities. However, during the maintenance program the EG alternated one session of physical fitness maintenance with other activities such as basketball and volleyball. No participant was allowed to carry out any physical fitness training outside of the supervised setting.Descriptive statistics (means and standard deviations) for age, body height, body mass index, and muscular and cardiovascular endurance results were calculated. The Student’s t test for independent samples was used to study the differences of the general characteristics between groups. As the BAH variable did not follow a normal distribution, the data was transformed using a logarithm . BecausAll students completed the development training program and 67 the maintenance training program according to previously established norms . Retest data of four participants from the EG and one from the CG were excluded due to missed classes in the maintenance training program and absence in the retest session test, respectively. The EG participants finally considered for analysis obtained an average attendance of 94% and 100% in the development and maintenance training program, respectively. The Student’s t for independent samples results did not show statistically significant differences in the general characteristics between EG and CG.Sit-ups in 30 seconds test. The EG had significantly greater gains in SUP compared to the CG   = 5.994; p = 0.003; η2p= 0.087; P = 0.875]. The EG participants significantly increased FAH from pretest to posttest (p = 0.009) and from pretest to retest (p < 0.001). For the EG, the improvement from the posttest to the retest approached statistical significance (p = 0.065). No differences were found for the CG (p ≥ 0.324). The test-retest reliability for the BAH was 0.95 (0.90–0.97).20-m endurance shuttle run test. The EG had significantly greater gains in ESR compared to the CG . The ANOVA with Bonferroni adjustment showed that the EG increased significantly from pretest to posttest (p = 0.015) though improvement from the pretest to the retest approached statistical significance (p = 0.088). No significant differences were found from posttest to retest for the EG (p = 0.210). No differences were found for CG (p ≥ 0.975). The test-retest reliability for the ESR was 0.90 (0.81–0.95).The results of the present study show that it is possible to develop both muscular and cardiovascular endurance by means of an eight-week circuit training program in the PE setting. Previous studies in which children performed an extra-curricular circuit training program confirmed a significant improvement on both muscular and cardiorespiratory fitness .Previous studies in which children performed an extra-curricular circuit training program confirmed a significant improvement on both muscular and cardiorespiratory fitness . NevertOne of the main objectives of the PE teachers at these educational levels is to make the pupils active as long as possible during the classes. With the circuits method the pupils can easily reach the minimum motor engagement time  at the One of the most important outcomes of this study was that a maintenance program carried out once a week in four weeks could be effective to maintain the gains previously obtained. As explained before, the majority of studies coincide in eight weeks setting as the period of inactivity determining the complete loss of previous physical fitness gains .In the present study the sum of the periods of detraining and maintenance was eight weeks, thus an unsatisfactory design of the maintenance program should have matched a decrease (or the complete dissipation) of the physical fitness profits. Nevertheless, results were positive since the muscular and cardiovascular endurance was maintained after these weeks.In line with the present study, In conclusion, the present study suggests that it is possible to develop and maintain muscular and cardiovascular endurance through a short-term program in the PE setting. Maintenance programs appear to be necessary in the school context to make the physical fitness training effective and feasible within a school plan, permitting at the same time the regular development of other curricular contents. Their utilization could permit the PE teacher to design programs that guarantee the maintenance of previous muscular and cardiovascular endurance gains in a few sessions. Even though more research is needed to confirm these results, the maintenance program could become a principal element to normal PE planning in the future. A limitation of the present study was the fact that the intervention program did not include many playful tasks. At these ages in PE classes it is important to develop contents mainly through ludic activities. Future interventions should focus on physical fitness programs based on stations with games, as well as the effect of the combination of different frequencies and durations of maintenance training.


Early and accurate diagnosis of dengue infection is essential for control of disease outbreaks. Recently, the dengue virus non-structural antigen 1 (NS1), a conserved and secreted glycoprotein, has been used as a marker for early diagnosis of dengue with convenience and cost-effectiveness. Serological tests of dengue IgM and IgG antibodies are still the most widely used for diagnosis of dengue. In order to assess combined diagnostic value of these tests, we study the kinetic profiles of circulating NS1, dengue IgM and IgG antibodies over the course of the disease by using an in-house dengue type 1 (DENV1) specific NS1 capture ELISA and the commercial Panbio Dengue IgM and IgG capture ELISAs.A panel of 313 acute-and early convalescent-phase serum specimens from 140 DENV1 primary infected patients during an outbreak of dengue in Guangzhou, China, in 2006 were studied. Dengue NS1 presented high levels in acute-phase serum samples. It was detectable as early as day 1 of illness, and up to 14 day after onset. The sensitivity of NS1 detection was ranged from 81.8% to 91.1% with samples taken during the first 7 days. Anti-dengue IgM antibody was detectable on the third day of onset with the positive rate of 42.9%, and rapidly increasing to 100% by day 8 of illness. Anti-dengue IgG antibody was detectable on the fifth day of onset with low level at the first week of onset, and slowly increasing to 100% by day 15 of illness. Combining the results of NS1 and IgM antibody detection allowed positive diagnosis in 96.9% -100% for samples taken after day 3 of onset.Dengue NS1 detection might shorten the window period by first few days of illness. A combination of dengue NS1 antigen and IgM antibody testing facilitates enhanced diagnosis rates. The procedures should be suitable for developing countries where dengue is endemic. The incnfection ,4. More nfection -12. NS1 nfection ,10.ELISA-based detection of viral antigens and specific antibodies have the advantage of being easier to perform and standardize, specially being suitable for resource poor countries. Consequently, these procedures are likely to become routine methods for diagnosing dengue infection. An understanding of the kinetic profiles of dengue NS1, as well as dengue IgM and IgG antibody responses will help clarify the advantages and disadvantages of these tests for diagnosing dengue infection. In this study, we used a well-characterized panel of acute and early convalescent-phase serum specimens collected from dengue patients during DENV1 outbreak in Guangzhou, China, in 2006 to study the kinetic profiles of circulating NS1, dengue IgM, and IgG antibody responses over the course of the disease. The aim of the present study was to evaluate combined diagnostic value of these tests.A panel of 313 acute- and convalescent-phase serum specimens were collected between days 1 and 27 after the onset of symptoms from 140 infected patients during the disease outbreak in Guangzhou, Guangdong province, China, in 2006 . Disease450 value of negative controls plus 5-times the SD. The result was considered positive if a sample yielded an OD450 value above the cutoff value.Detection of NS1 in the serum samples with an in-house DENV1 specific NS1 capture ELISA was performed according to the published protocol with minor modifications . BrieflyAnti-dengue IgM and IgG antibodies were measured with the commercially available Panbio Dengue IgM capture ELISA (Cat. No. EDEN01M) and Dengue IgG capture ELISA (Cat. No. E-DEN02G). The results are classified as positive, negative and equivocal according to the manufacturer's instructions. The initial equivocal result was retested to confirm the result.The infection status was determined by Panbio Dengue IgM and IgG capture ELISA as described above according to the manufacturer's instructions. According to the published criteria, the infection status was classified as follows: a serum positive for IgM antibody and negative for IgG antibody or a negative IgG test in a serum sample collected at least five days after disease onset, followed by seroconversion in the convalescent serum sample is considered as primary infections; a positive or negative serum for IgM antibody, but positive for IgG antibody test for an acute-phase sample obtained within 4 days of disease onset is considered as secondary infections -17.In this study, we analyzed 313 paired or multiple serum samples from 140 patients with laboratory confirmation of acute DENV1 infection by using the DENV1 NS1 ELISA, dengue IgM and IgG ELISA. All of these patients were classified as the primary infection based on the interpretative criteria described above. As shown in Figure The present study is the first report of kinetics of NS1, simultaneously, IgM and IgG responses over the course of the disease in DENV1 primary infected patients. A primary dengue infection has been characterized by a slow and low titer of IgG antibody response. IgM antibodies appear only 3 to 5 days after onset of the disease -4,18. ThThe levels of NS1 antigen might reflect the viral load during the course of disease as demonstrated by others . The NS1This study shows the kinetic profiles of circulating NS1, dengue IgM and IgG antibody responses as measured by ELISA-format assays of samples taken on different days after onset of symptoms. This work described here demonstrated that dengue NS1 antigen is a very promising early diagnostic marker. However, laboratory diagnosis must consider the timing of the clinical course, a strict diagnosis of acute dengue infections requires a combination of several tests performed at different stages of the disease.The authors declare that they have no competing interests.DH performed the NS1, IgM and IgG assays, analyzed the data and jointly drafted the manuscript. BD and MW collected serum samples and identified dengue infection by performed RT-PCR, virus isolation and serology. XD, YW, and KW jointly performed the NS1, IgM and IgG assays. YC and YP optimized the NS1 capture ELISA. XC conceived and designed the study, and drafted the manuscript. All authors read and approved the final manuscript.


Problem drinking, particularly risky single-occasion drinking (RSOD), also called “binge drinking”, is widespread among adolescents and young adults in most Western countries. Few studies have tested the effectiveness of interventions to reduce RSOD in young people with heterogeneous and particularly lower educational background.To test the appropriateness and initial effectiveness of a combined, individually tailored Web- and text messaging (SMS)–based intervention program to reduce problem drinking in vocational school students.The fully automated program provided: (1) online feedback about an individual’s drinking pattern compared to the drinking norms of an age- and gender-specific reference group, and (2) recurrent individualized SMS messages over a time period of 3 months. Generalized Estimating Equation (GEE) analyses were used to investigate the longitudinal courses of the following outcomes over the study period of 3 months: RSOD, alcohol-related problems, mean number of standard drinks per week, and maximum number of standard drinks on an occasion.P<.001), in the percentage of persons with alcohol-related problems , and in the mean number of standard drinks per week: 13.4 (SD 15.3) to 11.3 (SD 14.0), P=.002. They also revealed a trend toward a decrease in the mean of the maximum number of drinks consumed on an occasion: 11.3 (SD 10.3) to 10.5 (SD 10.3), P=.08.The program was tested in 36 school classes at 7 vocational schools in Switzerland. Regardless of their drinking behavior, 477 vocational school students who owned a mobile phone were invited to participate in the program. Of these, 364 (76.3%) participated in the program. During the intervention period, 23 out of 364 (6.3%) persons unsubscribed from participating in the program. The GEE analyses revealed decreases in the percentage of persons with RSOD from baseline  to follow-up assessment (67.6%, 188/278, The results show high acceptance and promising effectiveness of this interventional approach, which could be easily and economically implemented within school classes. Alcohol use is a major cause of the disease burden in most countries of the world . In EuroStudies testing the efficacy of interventions to reduce problem drinking in young people have been conducted predominantly in the United States and have been targeted toward college or university students . Within Social norm interventions provide information about the actual drinking norm in a reference group. They typically include an individualized drinking profile with the quantity of alcohol consumed in relation to peers, the money spent on alcohol, the calorie intake, and the individual risk patterns of alcohol-related negative consequences. In university and college students, normative feedback interventions delivered using the Web or computer, reduced drinking quantity, drinking frequency, and binge drinking in the short- and medium-term .Computer-tailored interventions based on the social norms approach are also promising for the reduction of problem drinking in populations with lower educational backgrounds. However, it should be noted that the efficacy of tailored messages depends on the individual’s ability and motivation to process information. The Elaboration Likelihood Model  posits tSocial norm interventions for reducing problem drinking typically consist of a single intervention session in which participants receive tailored Web or printed feedback. Due to their length of up to 7-8 pages of text and graphics, these interventions are primarily matched with persons of higher educational levels and higher NFCs. An approach that might be more effective for individuals with lower educational levels and that might also be more persuasive if persons are processing information on the peripheral route is to provide shorter, more recurrent feedback messages.Text messaging , which is available primarily via mobile phones, provides a suitable technology to deliver short and repeated feedback messages. This service allows a cost-effective instantaneous delivery of short messages directly to individuals at any time and place. In the field of alcohol prevention, SMS particularly allows the delivery of individualized messages at times when young people typically drink alcohol . In SwitSMS is increasingly being applied for behavior change interventions, particularly in smoking cessation and diabetes self-management ,21. For Vocational school students are typically characterized by heterogeneous educational levels, including a significant proportion with little or no educational attainment, and high prevalence rates of hazardous drinking . The objIn most European countries, vocational schools are post-secondary public schools that are analogous to American community colleges. They are part of the dual educational system that combines apprenticeships in a business context and vocational training in a school context. Vocational schools provide general education and specific skills for a particular profession.Based on data from the Swiss Federal Statistical Office, approximately half of all adolescents ages 16 to 19 currently attend vocational schools , with thA longitudinal pre-post study design was used to test the initial effectiveness of the program. Furthermore, differences in the longitudinal courses of the main outcome criteria between program participants and nonparticipants were explored.Directors or contact teachers for addiction prevention from all 23 vocational schools in the Swiss canton of Zurich were invited to participate through some of their classes in a study testing the effectiveness of a Web- and text messaging-based program to reduce problem drinking. Of these 23 schools, 7 vocational schools with a total of 36 school classes agreed to participate in the study. All vocational school students in the participating school classes were invited by externally trained staff to participate in an online health survey during a regular school lesson reserved for health education. To decrease reporting bias, the study assistants did not provide further information about the purpose of the study before the screening assessment was completed. The screening assessments were conducted between April 2012 and September 2012. At the time of the assessment, 490 students were present in the school classes, of whom 488 (99.6%) agreed to participate. The online screening included the assessment of demographic data, alcohol consumption, weekly physical activity, smoking status, and ownership of a mobile phone.The inclusion criterion for program participation was ownership of a mobile phone. A total of 477 of the 488 participants (97.7%) from the screening assessment owned a mobile phone. Subsequently, eligible persons were informed about the aim of the program, assessments, reimbursement, and data protection. Study and program information was provided online and in paper form by the study assistants. Eligible persons were informed that they could unsubscribe from program participation at any time simply by sending an SMS expressing their request to withdraw from the program. Additionally, they were informed that program participants would take part in a draw for 10 vouchers worth €50. Eligible persons could then decide whether to participate in the program or not. After providing informed consent online, all program participants were invited to choose a username and to provide their mobile phone number. Furthermore, additional alcohol-related variables were assessed.Follow-up assessments after 3 months were conducted in the participating school classes during regular school lessons using paper-and-pencil questionnaires. All vocational school students present in the school classes at the follow-up assessments  were invited to fill in a questionnaire.The study protocol was approved by the local Ethics Committee of the Canton of Zurich, Switzerland. The study was executed in compliance with the Declaration of Helsinki.The fully automated program was based on a LAMPP-system  and included an expert system that generated individually tailored online feedback and text messages. The interventional content was based on effective social norms intervention programs developed primarily for college and university students in the United States and Canada ,27 that The program, Alk-Check, automatically generated individually tailored online feedback and SMS messages using data from a comprehensive online assessment. The online assessment tool collected demographic information and information on alcohol consumption, drinking behavior , and alcohol-related problems. Age- and gender-specific norms for alcohol consumption were derived from a previous study  that assAfter completing the online assessment, individually tailored online feedback was provided. The online feedback was tailored according to the individual values on the following 4 baseline variables: gender, age, number of standard drinks in a typical week, and frequency of RSOD occasions in the last 30 days. The feedback included graphical and textual information concerning (1) drinks per week in relation to the age and gender-specific reference group, (2) financial costs of drinking, (3) calories consumed with alcoholic drinks, and (4) number of heavy drinking occasions in relation to the age and gender-specific reference group. The online feedback could be printed and sent by mail to the participants’ email accounts.On the first level, the content and number of text messages were tailored according to baseline drinking patterns. Participants were assigned to one of three risk groups , baseOn the second level, the content of the text messages was tailored according to the individual values on the following baseline variables: gender, motivation for reduced alcohol consumption, alcohol-related problems, typical drinking day and time, number of standard drinks in a typical week, and maximum number of drinks on a single occasion during the last 30 days.Participants from all risk groups received text messages for a period of 12 weeks. Participants of the non-risk group received one weekly text message providing information from the following content categories: (1) drinking and body weight/fitness, (2) resisting peer pressure, (3) pros of sensible drinking, and (4) motivation to maintain sensible drinking.Participants of the low-risk group received one weekly text message providing information from the following content categories: (1-3), (5) motivation for sensible drinking, (6) alcohol-related problems, (7) maximum number of drinks on a single occasion and related risks, (8) risks of binge drinking, and (9) importance of reducing alcohol consumption. Additionally, they received biweekly text messages sent on the individually indicated typical drinking day and time. The latter messages specifically focused on strategies to reduce alcohol consumption and to motivate them toward sensible drinking practices. Participants of the high-risk group received one weekly text message providing information from the content categories: (1-3), (5-9), and (10) local outpatient services for alcohol counseling. They also received the additional biweekly text messages sent on the individually indicated typical drinking day and time that focused on strategies to reduce alcohol consumption and to motivate them to adopt sensible drinking practices. Examples of these text messages are shown in Before the study, a prototype of this program was tested and evaluated in 3 focus groups. Within these focus groups, vocational school adolescents aged between 16 and 20 years evaluated the program flow, the layout and content of the online assessment and feedback, and the content of the text messages. The optimizations that resulted from these focus groups were integrated in the final program version.The screening assessment included the following demographic variables: gender, age, education, and migration background. Common Swiss levels of educational attainment were assessed: (1) none, (2) secondary school, (3) extended secondary school, and (4) technical or high school. We assessed the country of birth of both parents of the vocational school students to identify a potential migration background. Based on this information, persons were assigned to one of the following categories: (1) persons with neither parent born outside Switzerland, (2) persons with one parent born outside Switzerland, and (3) persons with both parents born outside Switzerland.Tobacco smoking was assessed using the question, “Do you currently smoke cigarettes or did you smoke in the past?” with the following response options: (1) I smoke cigarettes daily, (2) I smoke cigarettes occasionally, but not daily, (3) I smoked cigarettes in the past, but I do not smoke anymore, and (4) I have never smoked cigarettes or have smoked fewer than 100 cigarettes in my life. Current daily and occasional smokers (categories 1 and 2) were considered smokers. Self-reported moderate to vigorous physical activity was measured by a question derived from the Health Behavior in School Aged Children (HBSC) study : “OutsidThe following 3 alcohol-related variables were included in the screening assessment: (1) frequency of RSOD occasions in the last 30 days ; (2) quantity of alcohol consumption, as assessed by a 7-day drinking calendar similar to the Daily Drinking Questionnaire (DDQ) [In the program, we also assessed alcohol-related problems and the motivation to reduce their alcohol consumption among participants. Alcohol-related problems were assessed by a questionnaire derived from the European School Survey Project on Alcohol and Other Drugs (ESPAD) . The parTo obtain the number of program participants who unsubscribed from the program (program attrition), we analyzed the log files of the SMS system in which all incoming and outgoing text messages were recorded.At follow-up, we also assessed an aspect of the usage of the SMS messages by asking the participants whether they (1) read through the SMS feedback messages thoroughly, (2) took only a short look at the feedback messages, or (3) did not read the feedback messages. Using a yes/no question, we evaluated whether the times when participants received the SMS messages were appropriate. We assessed whether the number of received SMS messages was appropriate or whether the participants would have preferred less or more SMS messages. The program participants also indicated their approval of certain statements concerning different aspects of the SMS messages and the online feedback , using the response categories “rather yes” and “rather no”.Outcome criteria for the test of effectiveness of the intervention were: (1) RSOD in the last 30 days (yes/no), (2) frequent RSOD in the last 30 days (0-2 RSOD occasions vs >2 RSOD occasions), (3) number of standard drinks in a typical week, (4) maximum number of drinks on an occasion in the last 30 days, and (5) alcohol-related problems in the last 3 months (yes/no).To test for baseline differences between program participants and nonparticipants, chi-square tests for categorical variables and Mann-Whitney U-tests for continuous or ordinal variables were used. For the attrition analysis (program participants lost to follow-up), we also used chi-square tests for categorical variables and Mann-Whitney U-tests for continuous or ordinal variables.We used Generalized Estimating Equation (GEE) analyses to investigate the longitudinal course of the outcome criteria over the study period of 3 months. GEE is a repeated-measures regression model that takes into account the correlation of the repeated measures within a person . It is aWe used logistic GEE models for the binary outcome variables “RSOD” and “alcohol-related problems”, as well as GEE models for the count data of the variables “number of standard drinks in a typical week” and “maximum number of drinks on a single occasion”. Each GEE-model included the examined time variable (baseline vs follow-up assessment) as a predictor and an outcome variable as dependent variable.Given the clustered nature of the data (students within school classes), we computed robust variance estimators for all GEE analyses. An alpha level of 0.05 (2-tailed) was chosen for all statistical tests in this study. All analyses were performed using the Stata software package, version 10.Beyond the statistical examination of the longitudinal course of the outcome variables for program participants, we graphically explored the longitudinal course of the outcome variables for non-program participants in contrast to program participants. Due to a lack of statistical power, we did not use statistical tests for this comparison.U=17402.0, P<.001) and had a higher maximum number of drinks on a single occasion .Of the 477 persons who owned a mobile phone and were therefore eligible for study participation, 364 (76.3%) registered for program participation. 2=6.2, P=.01) and to have more frequent RSOD within the last month .After 3 months, 367 of the 477 (76.9%) students who were eligible for program participation completed follow-up assessment: program participants 280/364 (76.9%); nonparticipants 87/113 (77.0%). The attrition analysis showed that persons dropping out were significantly more likely to be smokers  program participants unsubscribed from participating in the program.Out of the 280 program participants who could be reached for follow-up assessment, we obtained data concerning the program use and evaluation from 234 to 269 persons, depending on the respective variable. These different frequencies are due to missing data, inconsistent data, or variables that were only assessed in some persons depending on their previous answers.Of the program participants, 254 out of 269 persons (94.4%) with valid data indicated that they regularly received the SMS messages. Of the 249 persons with valid data, 124 (49.8%) indicated that they “read the SMS messages thoroughly”; 111 persons (44.6%) reported that they “took a short look at the feedback messages”; and 14 persons (5.6%) chose the predefined response category, “I did not read the feedback messages”.The time when participants received the SMS messages was rated as appropriate by 75.4% of the program participants (196/260). The number of received SMS messages was rated as appropriate by 57.5% (149/259); 35.5% (92/259) would have preferred fewer; and 6.9% (18/259) would have preferred more SMS messages. P<.001). Considering only program participants with appropriate follow-up data, the percentage of program participants with at least one RSOD occasion in the last month was 75.5% (210/278) at baseline and 67.6% (188/278) at follow-up . Considering only program participants with follow-up data, the percentage of program participants with more than two RSOD occasions in the last month was 48.2% (134/278) at baseline and 41.0% (114/278) at follow-up . Considering only program participants with follow-up data, the mean number of standard drinks in a typical week was 13.4 (SD 15.3) at baseline and 11.3 (SD 14.0) at follow-up . Considering only program participants with follow-up data, the maximum number of drinks on an occasion was 11.3 (SD 10.3) at baseline and 10.5 (SD 10.3) at follow-up . Considering only program participants with follow-up data, the percentage of persons with one or more alcohol-related problems in the last 3 months was 20.4% (57/280) at baseline and 14.3% (40/280) at follow-up.The GEE analyses revealed a statistically significant decrease in the percentage of persons with one or more alcohol-related problems in the last 3 months from the baseline assessment to the follow-up assessment  a large percentage of vocational school students could be reached by the program, (2) the acceptance of the program was good, and (3) the program may reduce problematic alcohol consumption in young people with heterogeneous and primarily lower educational levels.The data of this study and other epidemiological studies ,33 showeParticipation in the program was relatively independent of gender, age, and immigration background, but participation rates were higher for persons with lower school educations and for persons with higher alcohol consumption. The latter finding shows that we were able to particularly reach the main target group of the intervention, namely young people with problem drinking.The overall acceptance of the intervention was good. Nearly all program participants (94%) stayed logged in until the end of the program lasting 3 months. The SMS messages were read by almost all program participants (94%), and both the SMS messages and the online feedback were comprehensible for almost all participants . Room for improvement was indicated in particular concerning the tailoring of the SMS messages. While nearly half of the participants (50%) rated the online feedback as individually tailored, only 34% indicated that they perceived the SMS messages as individually tailored.The results concerning the initial effectiveness of this program derived from a pre-post investigation are promising. The data revealed a statistically significant decrease in the percentage of persons with at least one RSOD occasion in the last month from baseline assessment (76%) to follow-up assessment (68%), as well as a statistically significant decrease in the percentage of persons with more than two RSOD occasions in the last month (from 48% to 41%). Furthermore, we found statistically significant decreases in the percentages of persons with alcohol-related problems and in the mean number of standard drinks per week. These positive changes could not be observed in persons not participating in the program.One limitation of this study is its lack of a control group that was derived on the basis of random assignment. Although participants were included into the study regardless of their drinking behavior, the comparison of baseline characteristics of program participants and nonparticipants indicates toward higher levels of alcohol consumption in program participants. Therefore, beyond the intervention effects, regression to the mean might have influenced the course of alcohol-related variables from baseline to follow-up. However, lack of a control group resulted in a greater proximity to prevention practices and allowed a better estimation of the participation rate in the program.This is the first study to test a combined Web- and SMS-based intervention program for the reduction of problem drinking and one of the very few studies to test an intervention reducing problem drinking in a school sample. The results of this study show appropriateness and promising effectiveness for this intervention approach of combining singular online feedback to provide comprehensive individualized data about a person’s alcohol consumption compared to an age- and gender-specific reference group and repeated individualized SMS messages encouraging sensible drinking. The intervention could be easily and economically implemented within school classes. Based on these initial positive results, testing this interventional approach within a randomized controlled trial would be reasonable.