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A recent systematic analysis showed that in 2011, 314 (296 - 331) million children younger than 5 years were mildly, moderately or severely stunted and 258 (240 - 274) million were mildly, moderately or severely underweight in the developing countries . In iran a study among 752 high school girls in sistan and baluchestan showed prevalence of 16.2%, 8.6% and 1.5%, for underweight, overweight and obesity, respectively . The prevalence of malnutrition among elementary school aged children in tehran varied from 6% to 16% . Anthropometric study of elementary school students in shiraz revealed that 16% of them suffer from malnutrition and low body weight . Snack should have 300 - 400 kcal energy and could provide 5 - 10 g of protein / day . Nowadays, school nutrition programs are running as the national programs, world - wide . National school lunch program in the united states there are also some reports regarding school feeding programs in developing countries . In vietnam, school base program showed an improvement in nutrient intakes . In iran a national free food program (nffp) is implemented in elementary schools of deprived areas to cover all poor students . However, this program is not conducted in slums and poor areas of the big cities so many malnourished children with low socio - economic situation are not covered by nffp . Although the rate of poverty in areas known as deprived is higher than other areas, many students in deprived areas are not actually poor and can afford food . Hence, nutritional value of the nffp is lower than the scientific recommended snacks for this age group . Furthermore, lack of variety of food packages has decreased the tendency of children toward nffp . On the other hand, the most important one is ministry of education (moe) of iran, which is responsible for selecting and providing the packages for targeted schools . The ministry of health (moh) is supervising the health situation of students and their health needs . Welfare organizations, along with charities, have the indirect effect on nutritional status of students by financial support of their family . Provincial governors have also the role of coordinating and supervising all activities of these organizations . Parent - teacher association is a community - based institution that participates in school's policy such as nffp . In addition to these organizations, nutritional literacy of students, their parents and teachers, is a very important issue, which could affect nutritional status of school age children . Therefore, the present study was conducted with the aim of improving the nffp, so that by its resources all poor children will be covered even in big cities . Moreover, all food packages were replaced by nutritious and diverse packages that were accessible for non - poor children . According to the aim of this study and multiple factors that could affect the problem, public health advocacy has been chosen as the best strategy to deal with this issue . Therefore, the present study determines the effects of nutrition intervention in an advocacy process model on the prevalence of underweight in school aged children in the poor area of shiraz, iran . This interventional study has been carried out between 2009 and 2010 in shiraz, iran . This survey was approved by the research committee of shiraz university of medical sciences . In coordination with education organization of fars province two elementary schools and one middle school in the third region of the urban area of shiraz were selected randomly . In those schools all students (2897, 7 - 13 years old) were screened based on their body mass index (bmi) by nutritionists . According to convenience method all students divided to two groups based on their economic situation; family revenue and head of household's job and nutrition situation; the first group were poor and malnourished students and the other group were well nourished or well - off students . For this report, the children's height and weight were entered into center for disease control and prevention (cdc) to calculate bmi and bmi - for - age z - scores based on cdc for diseases control and prevention and growth standards . The significance of the difference between proportions was calculated using two - tailed z - tests for independent proportions . For implementing the interventions, the advocacy process model weight was to the nearest 0.1 kg on a balance scale (model #seca scale). Standing height was measured to the nearest 0.1 cm with a wall - mounted stadiometer . Advocacy group formation: this step was started with stakeholder analysis and identifying the stakeholders . The team was formed with representatives of all stakeholders include; education organization, welfare organization, deputy for health of shiraz university, food and cosmetic product supervisory office and several non - governmental organizations and charities . Situation analysis: this was carried out by use of existing data such as formal report of organizations, literature review and focus group with experts . The prevalence of malnutrition and its related factors among students was determined and weaknesses and strengths of the nffp were analyzed . Accordingly, three sub - groups were established: research and evaluation, education and justification and executive group . Designing the strategies: three strategies were identified; education and justification campaign, nutritional intervention (providing nutritious, safe and diverse snacks) and networking . Performing the interventions: interventions that were implementing in selected schools were providing a diverse and nutritious snack package along with nutrition education for both groups while the first group (poor and malnourished students) was utilized the package free of charge . Education and justification intervention: regarding the literature review and expert opinion, an educational group affiliated with the advocacy team has prepared educational booklets about nutritional information for each level (degree). Accordingly, education of these booklets has been integrated into regular education of students and they educated and justified for better nutrition life - style . It leads the educational group to hold several meeting with the student's parents to justify them about the project and its benefit for their children . After these meetings, parental desire for participation in the project illustrated the effectiveness of the justification meeting with them . For educate fifteen talk show programs in tv and radio, 12 published papers in the local newspaper, have implemented to mobilize the community and gain their support . Healthy diet, the importance of breakfast and snack in adolescence, wrong food habits among school age children, role of the family to improve food habit of children were the main topics, in which media campaign has focused on . Nutritional intervention: the snack basket of the students was replaced with traditional, nutritious and diverse foods . In general, the new snack package in average has provided 380 kcal energy, 15 g protein along with sufficient calcium and iron . Low economic and malnourished children were supported by executive group affiliated with advocacy team and the rest of them prepare their snack by themselves . Research and evaluation: in this step, the literacy and anthropometric indices (bmi) of students were assessed before and after the interventions . The reference for anthropometric measures was the world health organization / national center for health statistics (who / nchs) standards and the cut - offs were - two standard deviations (sd) from the mean . Each student that was malnourished and poor has been taken into account for free food and nutritious snacks . Demographic information, height, weight and knowledge of the students were measured by use of a validated and reliable (cronbach's alpha was 0.61) questionnaire . This project is granted by shiraz university of medical sciences, charities and welfare organization and education organization of fars province . Statistical analyses were performed using the statistical package for the social sciences (spss) software, version 17.0 (spss inc ., the results are expressed as mean sd and proportions as appropriated . In order to determine the effective variables on the malnutrition status paired t test was used to compare the end values with baseline ones in each group . In this project, the who z - score cut - offs used were as follow: using bmi - for - age z - scores; overweight:> + 1 sd, i.e., z - score> 1 (equivalent to bmi 25 kg / m), obesity:> + 2 sd (equivalent to bmi 30 kg / m), thinness: <2 sd and severe thinness: <3 sd . This interventional study has been carried out between 2009 and 2010 in shiraz, iran . This survey was approved by the research committee of shiraz university of medical sciences . In coordination with education organization of fars province two elementary schools and one middle school in the third region of the urban area of shiraz were selected randomly . In those schools all students (2897, 7 - 13 years old) were screened based on their body mass index (bmi) by nutritionists . According to convenience method all students divided to two groups based on their economic situation; family revenue and head of household's job and nutrition situation; the first group were poor and malnourished students and the other group were well nourished or well - off students . For this report, the children's height and weight were entered into center for disease control and prevention (cdc) to calculate bmi and bmi - for - age z - scores based on cdc for diseases control and prevention and growth standards . The significance of the difference between proportions was calculated using two - tailed z - tests for independent proportions . For implementing the interventions, weight was to the nearest 0.1 kg on a balance scale (model #seca scale). Standing height was measured to the nearest 0.1 cm with a wall - mounted stadiometer . Advocacy group formation: this step was started with stakeholder analysis and identifying the stakeholders . The team was formed with representatives of all stakeholders include; education organization, welfare organization, deputy for health of shiraz university, food and cosmetic product supervisory office and several non - governmental organizations and charities . Situation analysis: this was carried out by use of existing data such as formal report of organizations, literature review and focus group with experts . The prevalence of malnutrition and its related factors among students was determined and weaknesses and strengths of the nffp were analyzed . Accordingly, three sub - groups were established: research and evaluation, education and justification and executive group . Designing the strategies: three strategies were identified; education and justification campaign, nutritional intervention (providing nutritious, safe and diverse snacks) and networking . Performing the interventions: interventions that were implementing in selected schools were providing a diverse and nutritious snack package along with nutrition education for both groups while the first group (poor and malnourished students) was utilized the package free of charge . Duration of intervention was 6 months . Education and justification intervention: regarding the literature review and expert opinion, an educational group affiliated with the advocacy team has prepared educational booklets about nutritional information for each level (degree). Accordingly, education of these booklets has been integrated into regular education of students and they educated and justified for better nutrition life - style . Obviously, student's families had remarkable effect on children's food habit . It leads the educational group to hold several meeting with the student's parents to justify them about the project and its benefit for their children . After these meetings, parental desire for participation in the project illustrated the effectiveness of the justification meeting with them . Educate fifteen talk show programs in tv and radio, 12 published papers in the local newspaper, have implemented to mobilize the community and gain their support . Healthy diet, the importance of breakfast and snack in adolescence, wrong food habits among school age children, role of the family to improve food habit of children were the main topics, in which media campaign has focused on . Nutritional intervention: the snack basket of the students was replaced with traditional, nutritious and diverse foods . In general, the new snack package in average has provided 380 kcal energy, 15 g protein along with sufficient calcium and iron . Low economic and malnourished children were supported by executive group affiliated with advocacy team and the rest of them prepare their snack by themselves . Research and evaluation: in this step, the literacy and anthropometric indices (bmi) of students were assessed before and after the interventions . The reference for anthropometric measures was the world health organization / national center for health statistics (who / nchs) standards and the cut - offs were - two standard deviations (sd) from the mean . Each student that was malnourished and poor has been taken into account for free food and nutritious snacks . Demographic information, height, weight and knowledge of the students were measured by use of a validated and reliable (cronbach's alpha was 0.61) questionnaire . This project is granted by shiraz university of medical sciences, charities and welfare organization and education organization of fars province . Advocacy group formation: this step was started with stakeholder analysis and identifying the stakeholders . The team was formed with representatives of all stakeholders include; education organization, welfare organization, deputy for health of shiraz university, food and cosmetic product supervisory office and several non - governmental organizations and charities . Situation analysis: this was carried out by use of existing data such as formal report of organizations, literature review and focus group with experts . The prevalence of malnutrition and its related factors among students was determined and weaknesses and strengths of the nffp were analyzed . Accordingly, three sub - groups were established: research and evaluation, education and justification and executive group . Designing the strategies: three strategies were identified; education and justification campaign, nutritional intervention (providing nutritious, safe and diverse snacks) and networking . Performing the interventions: interventions that were implementing in selected schools were providing a diverse and nutritious snack package along with nutrition education for both groups while the first group (poor and malnourished students) was utilized the package free of charge . Education and justification intervention: regarding the literature review and expert opinion, an educational group affiliated with the advocacy team has prepared educational booklets about nutritional information for each level (degree). Accordingly, education of these booklets has been integrated into regular education of students and they educated and justified for better nutrition life - style . Obviously, student's families had remarkable effect on children's food habit . It leads the educational group to hold several meeting with the student's parents to justify them about the project and its benefit for their children . After these meetings, parental desire for participation in the project illustrated the effectiveness of the justification meeting with them . Educate fifteen talk show programs in tv and radio, 12 published papers in the local newspaper, have implemented to mobilize the community and gain their support . Healthy diet, the importance of breakfast and snack in adolescence, wrong food habits among school age children, role of the family to improve food habit of children were the main topics, in which media campaign has focused on . Nutritional intervention: the snack basket of the students was replaced with traditional, nutritious and diverse foods . In general, the new snack package in average has provided 380 kcal energy, 15 g protein along with sufficient calcium and iron . Low economic and malnourished children were supported by executive group affiliated with advocacy team and the rest of them prepare their snack by themselves . Research and evaluation: in this step, the literacy and anthropometric indices (bmi) of students were assessed before and after the interventions . The reference for anthropometric measures was the world health organization / national center for health statistics (who / nchs) standards and the cut - offs were - two standard deviations (sd) from the mean . Each student that was malnourished and poor has been taken into account for free food and nutritious snacks . Demographic information, height, weight and knowledge of the students were measured by use of a validated and reliable (cronbach's alpha was 0.61) questionnaire . This project is granted by shiraz university of medical sciences, charities and welfare organization and education organization of fars province . Statistical analyses were performed using the statistical package for the social sciences (spss) software, version 17.0 (spss inc ., chicago, il, usa). The results are expressed as mean sd and proportions as appropriated . In order to determine the effective variables on the malnutrition status paired t test was used to compare the end values with baseline ones in each group . Two - sided p <0.05 was considered to be statistically significant . In this project, the who z - score cut - offs used were as follow: using bmi - for - age z - scores; overweight:> + 1 sd, i.e., z - score> 1 (equivalent to bmi 25 kg / m), obesity:> + 2 sd (equivalent to bmi 30 kg / m), thinness: <2 sd and severe thinness: <3 sd . Study population contains 2897 children; 70.8% were primary school students and 29.2% were secondary school students . 2336 (80.5%) out of total students were well - off and 561 children (19.5%) were indigent . 19.5% of subjects were in case group (n = 561) and 80.5% were in the control group (n = 2336). The mean of age in welfare group was 10.0 2.3 and 10.5 2.5 in non - welfare group . Demographic characteristics of school aged children in shiraz, iran table 2 shows the frequency of subjects in different categories of bmi for age in non - welfare and welfare groups of school aged children separately among boys and girls before and after a nutrition intervention based on advocacy process model in shiraz, iran . The frequency of subjects with bmi lower than <2 sd decreased significantly after intervention among non - welfare girls (p <0.01). However, there were no significant decreases in the frequency of subjects with bmi lower than <2 sd boys . When we assess the effect of intervention in total population without separating by sex groups, we found no significant change in this population [table 3]. Bmi for age for iranian students aged 7 - 14 years based on gender according to who growth standards 2007 bmi for age for iranian students aged 7 - 14 years according to who growth standards 2007 in non - welfare and welfare groups of total population table 4 has shown the prevalence of normal bmi, mild, moderate and severe malnutrition in non - welfare and welfare groups of school aged children separately among boys and girls before and after a nutrition intervention based on advocacy process model . According to this table there were no significant differences in the prevalence of mild, moderate and severe malnutrition among girls and boys . Table 4 also shows the mean of all anthropometric indices changed significantly after intervention both among girls and boys . The pre- and post - test education assessment in both groups showed that the student's average knowledge score has been significantly increased from 12.5 3.2 to 16.8 4.3 (p <0.0001). Bmi, height and weight in non - welfare and welfare groups of school aged children separately in males and females before and after a nutrition intervention based on advocacy process model in shiraz, iran according to study's finding the odds ratio (or) of sever thinness and thinness in non - welfare compared with welfare is 3.5 (or = 3.5, confidence interval [ci] = 2.5 - 3.9, p <0.001). Furthermore, the finding showed or of overweight and obesity in welfare compared to non - welfare is 19.3 (or = 19.3, ci = 2.5 - 3.9, p = 0.04). The result of this community intervention study revealed that nutrition intervention based on advocacy program had been successful to reduce the prevalence of underweight among poor girls . This study shows determinant factor of nutritional status of school age children was their socio - economic level . According to our knowledge, this is the first study, which determines the effect of a community intervention based on advocacy process on the malnutrition indices in a big city (shiraz) in iran . The other program in iran (nffp) is specified to deprived area and is not conducted in big cities . Allocating millions of dollars to nffp by government, selecting the malnourished students through an active screening system at primary and middle schools, paying attention of policy makers to student's nutrition have provided the opportunity to combat the problem . However, negligence of under - poverty line, providing poor snacks in terms of nutritional value and lack of variety are the main defects of this program . Advocacy by definition is a blending of science, ethics and politics for comprehensive approaching health issues . By using advocacy program in california among the high school students for improving their nutrition and physical activity angeles unified school district participants emphasized on nutrition classes for families as well as students in addition to other interventions . In the present study another study revealed that evaluability assessment gave stakeholders the opportunity to reflect on the project and its implementation issues . It seems that in iran, free food program among the students not only is needed in deprived areas, but also it should be performed in big cities such as shiraz . At baseline, no significant difference was founded among wealthy students between the pre- and post - nutritional status intervention . In contrast, the numbers of students who have malnutrition decreased from 44% to 39.4%, which was identified as a significant among impecunious girls students . There was also a significant increase in the proportion of children with bmi that was normal for age (2 to + 1 sd) most of the published community interventions showed better results among females compared with males . This difference in the impact of nutritional interventions between male and female might be related to the different age of puberty in the female population compared to the male population . In the age range of the present study female although, there is no nffp in big cities of iran, there are some programs for improving the nutritional status such as providing free milk in schools . A recent publication has shown that school feeding programs focus on milk supplementation had beneficial effects on the physical function and school performances specifically among girls in iran . The results of the mentioned study showed an improvement in the weight of children, psychological test's scores and the grade - point average following this school feeding program . The intervention in the present study had focused on the snack intake in the school time . There are some reports regarding the nutrition transition in iran, which shows the importance of nutrition intervention to provide more healthy eating dietary habits among welfare groups of adolescents . Hence, nutrition intervention especially in the form of nutrition education is needed in big cities and among welfare children and adolescents . Although a study among iranian adolescents showed that dietary behavior of adolescents does not accord to their knowledge, which emphasize on the necessity of community intervention programs . A recent study regarding the major dietary pattern among iranian children showed the presence of four major dietary patterns, in which fast food pattern and sweet pattern as two major dietary patterns can be mentioned among iranian children . In advocacy program audience's analysis accordingly, one of the prominent strategies in this study was working with media and was meeting with parent - teacher association that both of them were secondary target audiences . We also took into account policy makers in different levels, from national to local as primary audiences . Advocacy team had several meetings with management and planning organization at national level and education organization of the fars province as well as principal of the targeted schools . Providing nutritious snacks need contribution of private sector such as food industries or factories, but their benefits should be warranted . Another choice was community involvement; which can be achieved by female health volunteers who are working with the health system . Advocacy team by using the support of charities and female health volunteers could establish a local factory that produced student's snacks based on the new definition . However, there are some challenges on the way of expanding this program . Mass production of the proposed snacks according to different desires and cultures and getting involvement of food industries with respect to marketing issues is one of those challenges . Moreover, providing a supportive environment in order to change the food habits of the students and their parents among the wide range of the population require a sustainable and continuous inter - sector collaboration . Although in a limited number of schools, in our study, interventions and advocacy program was successful, expanding this model to another areas around the country depends on convincing the policy makers at national level . In this regard, advocacy team should prepare evidenced based profile and transitional planning to convince the policy makers for improving the rule and regulation of nffp . The same as this study in other studies have also emphasized that there must be efforts to strengthen the capacity within the schools to deal with the nutritional problems either overweight, obesity or malnutrition by using of educational and nutritional intervention . Assessing the dietary adherence is very important in nutrition intervention among population . As this population was children and adolescents we had a limitation in the blood sample collection to assess the subject's dietary adherence . Furthermore, this intervention was only focused on the intake of snack in school time and we did not have comprehensive information on the dietary intake of children and adolescents after school all over the day . The investigators propose further investigation in different areas of the country based on socio - cultural differences in order to make necessary modification and adapt this model to other areas . Regarding the nutritional needs of the school age children, provision of a good platform for implementing and expanding this efficient model to the whole country based upon the socio - economic situation of each region is advisable to the moh and the moe . Community nutrition intervention based on the advocacy process model is effective on reducing the prevalence of underweight specifically among female school aged children.

It occurs in more than 50% of patients and may reach 90% in certain types of cancers, especially in patients undergoing chemotherapy and/or radiation therapy.1 anemia is defined as an inadequate circulating level of hemoglobin (hb) (hb <12 g / dl) and may arise as a result of the underlying disease, bleeding, poor nutrition, chemotherapy, or radiation therapy . Preliminary studies suggest that survival and loco - regional control after radiation therapy, especially in head and neck cancers, may be compromised by anemia.24 anemia often worsens symptoms such as fatigue, weakness, and dyspnea, and thus may have a negative effect on quality of life (qol) and performance status in patients with cancer . Thus, to improve physical functioning, qol, and prognosis in patients with cancer, it would be reasonable to take a proactive approach in identifying populations who need treatment for cancer - associated anemia (caa) and provide timely management . Blood transfusion is an effective way to replace depleted hb within a short period, but the effect is, unfortunately, temporary and can cause serious adverse risks and increased mortality . In randomized clinical trials in patients with caa, erythropoiesis - stimulating agents (esas) produced significant increases in hb level, decreased transfusion requirements, and improved qol.57 however, 30%50% of patients do not respond to such agents . In addition, the use of esas often causes concern about severe adverse reactions.6,8 in several studies, esas were found to shorten overall survival time, or time to tumor progression in patients whose hb level reached more than 12 g / dl . These studies included patients with different primary cancers, such as breast, lung, head and neck, cervix, and lymphomas.911 the lack of response to erythropoietin stimulation in patients with cancer is partly attributed to the functional iron deficiency state, in which the high rate of erythropoiesis exceeds the delivery of usable iron, despite adequate iron stores.12 absolute iron deficiency, in contrast, occurs when iron delivery is impaired because iron stores are depleted (serum ferritin, <100 ng / ml; transferring saturation, <20%).13 hepcidin, a peptide hormone produced by the liver, is up - regulated in chronic inflammatory states including cancer . Hepcidin inhibits iron transport across cell membranes, thus decreasing the accessibility of stored iron and gastrointestinal absorption of dietary iron, leading to an increased frequency of iron - restricted erythropoiesis.1416 many randomized trials examined the role of intravenous (iv) iron in addition to esas in the treatment of anemia in patients with cancer . Many of these studies showed improvement in esa response, time to maximal response, reduction in esa dose, and improvement in qol parameters (when measured) in favor of the combination over esas alone . The observed benefit was independent of baseline iron parameters.1721 one study found a 36% reduction in the number of patients transfused.21 this pilot study assessed the efficacy and feasibility of iv iron monotherapy in patients with cancer who have anemia and who are undergoing treatment with chemotherapy and/or radiation therapy without the use of esas . Patients received the study treatment for 12 weeks followed by a 4-week follow - up period . Eligible patients were at least 18 years old, about to start a cycle of chemotherapy and/or radiation therapy within 1 week of inclusion, and had a nonmyeloid malignancy, hb levels of 11.0 g / dl or less, a life expectancy of more than 24 weeks, and an eastern cooperative oncology group performance status of 02 . Patients were also required to have a serum ferritin level of 100 ng / ml or higher or transferrin saturation (tsat) levels of 15% or higher and to have received no esas or iv iron therapy within 30 days and no oral iron therapy (27 mg / day or more) within 7 days before enrollment . Patients were excluded for leukoerythroblastic features on blood film, hemolysis, gastrointestinal bleeding, folate or vitamin b12 deficiency, elevated serum ferritin (900 ng / ml) or transferrin saturation (tsat) (35%) levels, pregnancy or lactation, liver dysfunction (grade 2 or higher based on national cancer institute common toxicity criteria), renal dysfunction (serum creatinine levels 2.0 mg / dl), active infection requiring systemic antibiotics, personal or family history of hemochromatosis, comorbidities precluding study participation, hypersensitivity to iv iron, red blood cell transfusion within the last 2 weeks, or any investigational agent within 30 days before enrollment . Patients were not allowed to take any vitamin, mineral, or herbal supplements containing 27 mg or more of iron per day or 100 mg vitamin c per day during the study or follow - up period . Blood transfusions were permitted at the primary physician s discretion if hb levels decreased to 8 g / dl or less, and such patients were considered treatment failures . Written informed consent was provided by all patients before study participation, and the protocol and supporting documents were approved by the institutional review board of king hussein cancer center . The study was conducted in accordance with the declaration of helsinki and good clinical practice as contained in the us code of federal regulations that governs the protection of human subjects and the obligations of clinical investigators . Patients received 200 mg ferric hydroxide sucrose diluted in 100 ml normal saline and infused over the course of 1 hour weekly for a total of 12 weeks . The first dose was given during the first clinic visit (4 days from the initiation of chemotherapy or radiation therapy). Tsat was monitored, as protocol mandated withholding iron therapy when tsat levels were higher than 50% . At the first clinic visit (week 1; baseline), a blood sample was obtained for laboratory assessments before the study treatment was started . Patients attended weekly clinic visits for treatment and assessment; and returned for follow - up visits at week 14 which included a complete physical examination . Complete blood count and tsat were done every 3 weeks, and again 2 weeks after last treatment (week 14). Complete laboratory assessment (hb, serum ferritin, reticulocyte count, transferrin, tsat, serum iron, total iron binding capacity, red cell indices, white blood cell count with differential, platelet count, and serum chemistries) were done at week 1 and at week 14 (end of study). Adverse events were assessed at each clinic visit until study completion or withdrawal, and during the 30 days after the last study treatment . Hb test results were presented as mean, median, and range through all 12 weeks . Comparison between means of hb level were made between the baseline hb and hb levels in the following weeks, using t - test . A significance criterion of p <0.05 was used in the analysis . All analyses were performed using sas version 9.1 (sas institute inc, cary, nc, usa). Patients received the study treatment for 12 weeks followed by a 4-week follow - up period . Eligible patients were at least 18 years old, about to start a cycle of chemotherapy and/or radiation therapy within 1 week of inclusion, and had a nonmyeloid malignancy, hb levels of 11.0 g / dl or less, a life expectancy of more than 24 weeks, and an eastern cooperative oncology group performance status of 02 . Patients were also required to have a serum ferritin level of 100 ng / ml or higher or transferrin saturation (tsat) levels of 15% or higher and to have received no esas or iv iron therapy within 30 days and no oral iron therapy (27 mg / day or more) within 7 days before enrollment . Patients were excluded for leukoerythroblastic features on blood film, hemolysis, gastrointestinal bleeding, folate or vitamin b12 deficiency, elevated serum ferritin (900 ng / ml) or transferrin saturation (tsat) (35%) levels, pregnancy or lactation, liver dysfunction (grade 2 or higher based on national cancer institute common toxicity criteria), renal dysfunction (serum creatinine levels 2.0 mg / dl), active infection requiring systemic antibiotics, personal or family history of hemochromatosis, comorbidities precluding study participation, hypersensitivity to iv iron, red blood cell transfusion within the last 2 weeks, or any investigational agent within 30 days before enrollment . Patients were not allowed to take any vitamin, mineral, or herbal supplements containing 27 mg or more of iron per day or 100 mg vitamin c per day during the study or follow - up period . Blood transfusions were permitted at the primary physician s discretion if hb levels decreased to 8 g / dl or less, and such patients were considered treatment failures . Written informed consent was provided by all patients before study participation, and the protocol and supporting documents were approved by the institutional review board of king hussein cancer center . The study was conducted in accordance with the declaration of helsinki and good clinical practice as contained in the us code of federal regulations that governs the protection of human subjects and the obligations of clinical investigators . Patients received 200 mg ferric hydroxide sucrose diluted in 100 ml normal saline and infused over the course of 1 hour weekly for a total of 12 weeks . The first dose was given during the first clinic visit (4 days from the initiation of chemotherapy or radiation therapy). Tsat was monitored, as protocol mandated withholding iron therapy when tsat levels were higher than 50% . At the first clinic visit (week 1; baseline), a blood sample was obtained for laboratory assessments before the study treatment was started . Patients attended weekly clinic visits for treatment and assessment; and returned for follow - up visits at week 14 which included a complete physical examination . Complete blood count and tsat were done every 3 weeks, and again 2 weeks after last treatment (week 14). Complete laboratory assessment (hb, serum ferritin, reticulocyte count, transferrin, tsat, serum iron, total iron binding capacity, red cell indices, white blood cell count with differential, platelet count, and serum chemistries) were done at week 1 and at week 14 (end of study). Adverse events were assessed at each clinic visit until study completion or withdrawal, and during the 30 days after the last study treatment . Hb test results were presented as mean, median, and range through all 12 weeks . Comparison between means of hb level were made between the baseline hb and hb levels in the following weeks, using t - test . A significance criterion of p <0.05 was used in the analysis . All analyses were performed using sas version 9.1 (sas institute inc, cary, nc, usa). Twenty - five patients (17 women and 8 men) were eligible, consented, and included in the study; their mean age (standard deviation, [sd]) was 56 years (13.0 years). Chemotherapy varied according to the primary cancer and included anthracycline, platinum, taxanes, cyclophosphamide, high - dose ifosfamide, vincristine, vinblastine, bleomycin, and others . Many of the included patients had their chemotherapy treatment as second- or third - line therapy . Patients characteristics, including age, primary tumor, and active anticancer treatment are summarized in table 1 . One patient died during the study from his tumor (after week 2), and five patients withdrew from the study because of inconvenience (three after week 3, and two after week 4). Nineteen (76.0%) patients completed a minimum of three treatments, 15 (60.0%) completed nine treatments, and 14 (56.0%) completed all twelve planned weekly treatments . As seen in table 2, the mean hb level of the 25 patients at baseline was 9.6 g / dl (median, 9.9 g / dl; range, 6.9 g / dl10.9 g / dl). For the 15 patients who completed at least nine treatments, the mean change in their hb level was 1.7 g / dl (median, 1.1 g / dl; range, 1.9 g / dl to 3.2 g / dl). For the 14 patients who completed the whole treatment period (12 weeks), the mean hb level change was 2.1 g / dl (median, 1.3 g / dl; range, 0.2 g / dl to 4.6 g / dl; p = 0.0007). Eight (42.1%) of the 19 patients who completed at least three iron infusions had a more than 1 g / dl increase in their hb level . Hemoglobin level changes for the 14 patients who completed twelve iron infusions are shown in figure 1 . No iv iron - related adverse events were reported among patients during the study or the follow - up period . Tsat was monitored during the study period, and no patients had tsat levels increase to more than 50% . The highest ferritin level among patients who completed at least nine iv iron treatments was 1,170 ng / ml; the mean level at the end of study period for the whole group was 379 ng / ml . Five (20.0%) patients received blood transfusions and were considered treatment failures (three after week 3, transfused at hb levels of 6.9 g / dl, 7.8 g / dl, and 5.4 g / dl; one after week 4, transfused at an hb level of 8.2 g / dl; and one after week 9, transfused at an hb level of 7.2 g / dl). Low hb levels are associated with diminished qol and possibly decreased overall survival.2 successful treatment of anemia has undeniable benefits for patients, often yielding dramatic symptomatic improvement . Although the role of esas is well - established in treating caa, big concerns were recently raised about the negative effect of esas on survival in some patients with cancer.911 concerns about the risk for thromboembolism in patients with cancer with higher hb levels who are receiving esa were also addressed in many trials.22,23 in addition, the possible immunosuppressive effects of blood product transfusions that may have relevance to neoplasia progression were addressed before.24, 25 in our pilot study, we tested the feasibility of using iron supplementation alone to treat anemia in patients with cancer who are undergoing chemotherapy without the use of esas or blood transfusion, which could be a valid alternative, especially for patients with curable cancers . Oral iron is easier to administer and relatively inexpensive, but low patient adherence, poor enteral absorption, and poor tolerance because of a wide range of troublesome gastrointestinal adverse effects limit its overall effectiveness.26 anemia of chronic disease may occur in patients with cancer and is associated with an increase in hepcidin levels, which decreases oral iron absorption and bone marrow iron use, negating any possible effect of regular doses of oral iron.15 iv iron therapy significantly improves response to epoetin alfa when compared with oral iron or no iron in anemic patients with cancer who are receiving chemotherapy.1721 oral iron supplements with esas showed no significant benefit over esas alone in treating caa.21 sodium ferric gluconate and iron sucrose appear to have more favorable safety profiles over iron dextran . A large prospective safety comparison trial failed to show serious anaphylactoid reactions,27 which is confirmed in our study, in which no patients developed reactions and no patients withdrew from the study because of adverse effects . Given that the mean hb increase using esas with iv iron in one large controlled trial was 2.4 g / dl,21 the results obtained in our study are clinically significant . These findings should be further confirmed and better assessed in larger studies, in which questions such as the optimal timing of iv iron therapy with respect to chemotherapy and the optimal total dose of iv iron should be determined . The use of iv iron monotherapy was recently reviewed by a group in germany that studied the use of ferric carboxymaltose to replace esa and blood transfusions as a treatment for caa . Iron - deficient patients treated with ferric carboxymaltose alone (n = 233) had a median of 1.4 g / dl increase in hemoglobin levels compared with those receiving additional treatment with esas (n = 46; median, 1.6 g / dl). Our study, however, is peculiar in using iron therapy in a non - iron - deficiency state.28 iron overload after iv iron therapy, with potential concerns about the risk of developing secondary cancers and infection, might be raised . The highest serum ferritin level in the present study in patients who completed at least 9 weeks of iv iron therapy was 1,170 ng / ml . Most of the literature addressing cancer and infections in iron - overloaded patients comes from patients with hemochromatosis or patients who are undergoing hemodialysis . Published reviews report an increase in hepatocellular carcinoma only in patients with hemochromatosis after they develop cirrhosis.29 similarly data supporting the association between iv iron therapy and higher infection rate are weak and not well - supported.30 in fact, anemia itself is a risk factor for infections in patients receiving hemodialysis.31 a multivariate analysis of associations between iron and mortality in more than 58,000 patients receiving hemodialysis reported no increased death rate from serum ferritin levels as high as 1,200 ng / ml.30 the increasing cost of therapy in patients with cancer is of grave concern, which could be an additional benefit of iv iron over the use of esas in such patients . To further address many of the questions raised, our team is planning a bigger trial for iv iron in patients with cancer who have anemia to confirm the results discussed in this pilot trial . In addition, we will be looking into predictors of response to iv iron, such as serum hepcidin level . Iv iron therapy alone is safe and may be effective in improving hb levels in patients with cancer who are undergoing active anticancer therapy . Further randomized trials are needed to address many of the questions raised in our pilot study.

Tardive dystonia (td), a rarer side effect after longer exposure to antipsychotics, is characterized by local or general, sustained, involuntary contraction of a muscle or muscle group, with twisting movements, generally slow, which may affect the limbs, trunk, neck, or face . Td has been shown to develop in about 3% of patients who have had long - term exposure to antipsychotics . . The low risk of td for atypical antipsychotics is thought to result from their weak affinity for dopamine receptors . Compared with typical, atypical antipsychotic agents have a greater affinity for serotonin 5-ht2a than dopamine d2 receptors, with a low propensity to induce td . Among this olanzapine is thought to have preferential action at mesolimbic over nigrostriatal dopaminergic pathways and is, therefore, associated with a very low incidence of extrapyramidal symptom (eps). Furthermore, a retrospective analysis of controlled multicentric trials suggested that olanzapine also improves preexisting symptoms of tardive movements . We report a case of 20-year - old male, belonging to lower socioeconomic class, educated up to 2 standard, presented with severe unilateral dystonic left sided neck movements [figure 1]. Careful history exploration revealed he was taking risperidone 2 mg irregularly for 2 months and then olanzapine 5 mg for another 4 months . Picture of neck dystonia of patient at 19 years, the patient presented with occasional anger outbursts, getting provoked on small matters and beating family members, running away from home, screaming episodes occasionally, fearfulness, sleep disturbance for 2 days; which was precipitated after fever . According to the mother, one friend might have threatened / made fun of him actually and after that patient stopped going out of house, and displayed above mentioned symptoms . This was interpreted as psychosis with persecutory ideas, and he was treated with risperidone 2 mg / day for 2 months and then with olanzapine 5 mg / day for 4 months . In last two follow - ups patient did not present himself, and mother reported unusual neck movements, which were taken as a part of his overall psychopathology and not taken seriously, slight intermittent neck movements reported were missed as part of adolescent behavior problems mimicking some hero in movies . As neck dystonia increased, the patient had a severe disability as patient had to keep his hands behind his head for the support . The movement would decrease when the patient was lying down and was absent during sleep . He even stopped taking food due to severe neck movements making chewing and swallowing difficult . His birth and early developmental milestones were normal . During 210 years of age patient was inattentive and mildly hyperactive . Other siblings were educated with master's degree, and patient was also sent to school, but due to inattention and restlessness, he did not pass 2 standard after three attempts . He left the schooling . With average executive functioning and life skills, he worked as an unskilled laborer in the neighborhood shops as a helping hand ., he was found to be getting over familiar, cheerful, moody, and short tempered . Sometimes, the patient had inappropriate social judgment; for which his friends made fun of him, and teased him . On mental status assessment, routine investigations, thyroid function tests, electroencephalogram, fundus examination, cervical x - ray, magnetic resonance imaging brain were normal . After consulting neurophysician, wilson's disease and other secondary causes of dystonia were ruled out . The patient was treated with clonazepam 1 mg total dissolved solid (tds), tetrabenazine 25 mg tds, trihexiphenidyl 2 mg bipolar disorder (bd). After 2 months, there was some improvement of around 30% . Baclofen 10 mg was added; increased up to 20 mg, trihexyphenidyl reduced to 2 mg . With little improvement after 4 months of treatment for dystonia, levodopa + carbidopa (100 + 25) was added by neurophysician and increased up to tablet tds and baclofen omitted . After 12 months of treatment, patient has improved around 90% with tetrabenazine 75 mg, levodopa + carbidopa (100 + 25) - tablet bd, and clonazepam 1 mg bd . Earlier case reports reported td developing with high - dose atypical antipsychotics such as olanzapine 20 mg or aripiprazole 15 mg with longer duration of exposure of around 1215 months in established psychiatric illness like schizophrenia or any other psychotic illness . Eps in general and tardive dyskinesia, in particular, have been extensively studied in schizophrenia . Even though a number of studies suggest that bipolar patients experience higher rates of eps (parkinsonism, dystonia, akathisia) and td compared to patients with a diagnosis of schizophrenia, research within the bd population has been limited . The risk is found to be 3 to 5 times higher in elderly patients compared to young patients . In addition to age, the risk is directly proportional to: female gender, daily and total dose of the antipsychotic drug, presence of mood disorder, the use of anticholinergics with neuroleptics, previous physical therapies (electroconvulsive therapy), the presence of other physical illness such as diabetes or an organic disorder, younger age of exposure, and the presence of extrapyramidal symptoms early in treatment . This patient's severe dystonic neck movements developed within short period of 6 months of exposure to atypical antipsychotics risperidone 2 mg and then olanzapine 5 mg only, which can cause minimal extrapyramidal side effects . In this case, risk factors for developing serious disabling td were neuroleptic exposure, borderline intellectual functioning, externalizing behavior, probable misdiagnosis, and overlooking early indicators of side effects . This case highlights dangers of casually prescribing low dose second generation antipsychotics in patient with hyperthymic temperament and borderline intellectual functioning with vague short lasting presenting complaints; probably misdiagnosed as psychosis; leading to such severe adverse effects because patients with organic brain damage are more prone to develop adverse effects like td . Thus, judicious use of antipsychotics, with detailed and frequent assessments is important, and emergent stereotyped behavior or unexplained movements must be examined carefully and taken seriously . The authors certify that they have obtained all appropriate patient consent forms . In the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed . The authors certify that they have obtained all appropriate patient consent forms . In the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Lepidoptera include agricultural pests that, through feeding and other activities, negatively affect stored grains, food and fiber crops [2, 3]. Since a single lepidoptera adult can produce hundreds of eggs, and their primary food source is typically plant material, they can cause significant damage to agricultural crops . Although biological agents can help manage these insect pests, insecticides currently are essential for large - scale effective and economical pest control . These insecticides can also affect non - target organisms, including pollinators, and their application not only disrupts natural ecosystems but also reduces yields of crops that rely on pollination [5, 6]. The non - target effect of some pesticides is in part due to their effects on insect immunity, which is necessary for insect survival in natural environments . For example, currently used pesticides have been shown to affect cellular [710] and humoral [11, 12] immune responses and interfere with grooming behavior [13, 14]. These effects on immunity are likely non - specific and negatively impact the health of both the target pest and beneficial arthropods . Therefore, there is a need for novel target - specific approaches to control insect pests without affecting beneficial arthropods . Although immune pathways can be generally and non - specifically inhibited by pesticides, they also are a likely source of candidate molecules that could be inhibited for target - specific insect control since multiple classes of insect immunity genes, including signaling pathways, can be under strong selection for diversification . Fundamental mechanisms of innate immunity comprising cellular and humoral pathways are conserved throughout the animal kingdom and are controlled by signaling pathways activated by various stimuli [17, 18], including pathogen recognition by immune surveillance systems . Despite this overall conservation, aspects of immune systems are subject to strong selection to evolve in response to varying pathogen exposure and to pathogen evolution of virulence determinants that modulate immunity [15, 1921]. Such co - evolutionary dynamics can promote diversification of conserved elements of immunity as well as the recruitment of novel effectors . As such, the investigation of insect immune pathways and mechanisms of pathogen modulation can yield insights into components that may be susceptible to inhibition . For example, the insect pathogen xenorhabdus nematophila suppresses cellular and humoral immunity in the lepidopteran moths manduca sexta and spodoptera exigua [23, 24] but not in the dipteran fly drosophila melanogaster, suggesting the stage of immunity suppressed by x. nematophila may be absent from d. melanogaster, but present in lepidoptera . Since dipteran flies serve as pollinators [26, 27], decomposers, food sources for other animals, and pest control agents, capitalizing on the possible differences between dipteran and lepidopteran immune signaling cascades will help in the identification of targets for pest - specific inhibition . With this knowledge in hand, pest management can be achieved by developing small molecule inhibitors of these targets that will suppress pest insect immunity and lead to increased susceptibility to environmental pathogens . Indeed, many insecticides may contribute to insect (target and non - target) death by modulating aspects of immunity . The feasibility of targeted pest control via insect immune inhibition has been established for termites; a small molecule inhibitor of an immune surveillance protein led to faster termite death upon exposure to various pathogens . Much of our current knowledge of insect immune signaling pathways and receptor and effector function is based on the premiere model organism d. melanogaster, for which there are extensive genetic tools and several fully sequenced genomes . Well - established lepidopteran insect models such as the silkworm bombyx mori and the tobacco hornworm m. sexta also have been widely used to study insect immunity . These organisms have been particularly useful for investigating hemolymph proteins and hemocyte function because of their relatively large larval size and hemolymph volume . Many insects in the order lepidoptera are easy to rear in laboratory conditions, and new tools such as rna interference have been implemented successfully to study genetics of their immune systems [30, 31]. Also, their immune signaling pathways are gradually being revealed by genomic and transcriptomic data [3238]. Based on these model insect systems a fairly detailed picture of immunity, from pathogen detection to effector function, is emerging, though many gaps remain, particularly with regard to components that are unique to different insect orders . Here we review aspects of insect immunity with an emphasis on the similarities and distinctions between d. melanogaster and representative lepidoptera . In insects, the cellular immune response includes phagocytosis, nodulation and encapsulation [3942] and the humoral response involves the expression of antimicrobial peptides (amps) [43, 44] as well as the pro - phenol oxidase (propo) proteolytic cascade that results in formation of melanized nodules and toxic reactive compounds [45, 46]. Amps are small cationic peptides that insert into and disrupt microbial membranes, thereby killing and clearing pathogens . They are synthesized by hemocytes and to a greater extent in fat body from which they are released into the insect hemolymph rapidly after microbial infection [43, 47]. Amps are also expressed in extra - embryonic tissues of eggs, which may help protect the developing embryo from infection . Amps are a conserved component of immunity in plants and animals and while they have diverse structures most can be assigned to larger families such as cecropins, attacins, defensins and diptericins . Their diversity and immune effector function as well as their variant representation among insects (table 1) have made them a central focus in the study of invertebrate pathology [30, 52]. In d. melanogaster amp synthesis each of these pathways is activated by detection of microbial components via different pattern recognition receptors (prrs) that trigger, through complex regulatory cascades, nuclear factor kappa b (nf-b) dependent transcription of the genes encoding amps . After amps are translated in the cytoplasm they are released into the hemolymph where their high concentrations and broad activity are thought to enhance clearance of invading microorganisms from the insect . Bioinformatic and experimental data support the existence of the amp - inducing toll and imd pathways in lepidopterans, though not all components have been identified in model organisms such as m. sexta [32, 35]. The conserved presence of amps in immunity coupled with the possibility that certain elements of their induction pathways may vary among insects enhances the probability that microbially - induced amp expression could be inhibited in a pest - specific manner . As such, for the remainder of this review we focus on pathways leading to amp gene expression . In d. melanogaster, transcription of amp - encoding genes is activated by the nf-b family transcription factors in response to infection [6569] with distinct nf-b family transcription factors responding to the toll and immune deficiency (imd) signal transduction pathways . In response to toll pathway activation, the nf-b inhibitor cactus is phosphorylated and degraded allowing its targets, the nf-b factors dif and dorsal, to be translocated to the nucleus . Imd pathway activity culminates in the nf-b factor relish being activated by a stimulus - induced proteolytic cleavage . In the case of dif and dorsal, gram - positive bacterial and fungal infections primarily serve as the stimuli that induce degradation of cactus through the toll signaling pathway . In general, gram - negative bacterial infections of d. melanogaster stimulate the proteolytic cleavage of relish through the imd pathway . Once in the nucleus, these transcription factors drive the transcription of immune effectors, including amp genes whose promoters contain nf-b binding sites [70, 72]. Overall, nf-b proteins and their dna - binding specificities are conserved among organisms, including those lepidoptera studied to date [73, 74]. However, the nfb - binding regions for inhibitor of b (ib) proteins (e.g., cactus) are not conserved, suggesting diversification and co - adaptation between ib and nfb pairs . Also, recent work indicates that nf-b nuclear co - regulators may contribute to species - specific regulation of amp gene expression . Therefore, modulation of inhibitors and nuclear - co - regulators of nf-b - dependent transcription may be one avenue by which target - specific immune suppression could be achieved . In d. melanogaster, nf-b - dependent amp induction through the toll and imd pathways is activated by detection of microbial components via different pattern recognition receptors (prrs). Prrs are soluble or membrane - bound proteins that bind to specific microbe associated molecular patterns (mamps) such as lipopolysaccharide (lps), lipoteichoic acid (lta), peptidoglycan (pgn) or -1,3-glucan that are released from or are found on the cell surfaces of bacteria or fungi . Upon interaction with mamps, prrs can directly agglutinate pathogens or trigger proteolytic signaling cascades and cytokine release, which in turn lead to the activation of downstream cellular and humoral pathways, including pro - po activation and amp gene expression [16, 65, 76]. Pgn recognition proteins (pgrps) and -1,3-glucanase - related proteins (grps) were discovered in the lepidopteran silkworm (b. mori) by assaying for plasma components that activate the propo cascade . Pgrps were subsequently shown to be conserved across mammals and insects, and in d. melanogaster their role in the induction of amp gene expression through toll and imd pathways in response to pgn has been well documented [7983]. Similarly, grps have been shown to induce amp expression through toll pathway in response to fungal infections [79, 84]. In contrast, there is a dearth of literature linking specific pgrps or grps to amp induction in lepidoptera . Such a link is possible, since pgn and -1,3-glucan can activate amp gene expression in m. sexta and b. mori [8590] and multiple infection - induced pgrp- and grp - encoding genes have been identified in lepidoptera [32, 38, 54, 55, 9194]. However, there are numerous hints that lepidoptera and diptera may have evolved divergent mechanisms of linking pathogen detection to conserved toll and imd signal transduction cascades . First, a genome comparison between b. mori and d. melanogaster failed to identify 1:1 pgrp orthologs . Similarly, b. mori gram - negative binding protein (gnbp) and m. sexta microbe binding protein (mbp), members of the -1,3-glucanase - related protein superfamily [76, 95], appear to be distantly related to d. melanogaster gnbps, suggesting divergence of this group of proteins . M. sexta mbp expression is strongly up - regulated in fat body after immune challenge and shows specific binding to lta, lps, dap - pgn . Also, in contrast to the situation in d. melanogaster, highly purified lps and lta are inducers of amp gene expression in lepidoptera, though not as potently as crude lps (with contaminating pgn) or purified pgn [85, 90, 96, 97]. This raises the possibility that different mamps or combinations of mamps are most efficacious in eliciting amp gene expression in lepidoptera relative to diptera . Also, since purified lps can elicit amp expression in lepidoptera but not d. melanogaster, lepidoptera have either a distinct repertoire of prrs responsible for lps - dependent triggering of imd or toll pathways, or an as - yet undiscovered pathway that links lps to amp induction . Testing these ideas awaits the identification of the suite of prrs and signal transduction pathways responsible for transducing lps, lta, pgn, or combinatorial microbial signals to amp gene expression . One class of lepidopteran prr that may mediate infection - dependent induction of amps is the c - type lectins (ctls), ca - dependent, secreted proteins that have carbohydrate - binding capabilities . Similar to some ctls of d. melanogaster, several ctls of m. sexta and b. mori [54, 99] are reported to mediate induction of cellular responses and the propo cascade . Although the nomenclature quickly becomes confusing, ctls include lipopolysaccharide - binding protein (lbp). M. sexta iml-1 binds to gram - positive and gram - negative bacteria as well as yeast, iml-2 shows specific binding to lps, iml-3 and iml-4 show specific binding to lps and lta, and iml-3 can also bind laminarin, a -1,3-glucan [103, 104]. Diversity in ctl carbohydrate - binding specificities may result in lineage - specific pathogen recognition - signal transduction connections . Of particular relevance to the theme of this review are prrs present in lepidoptera but not diptera (table 1). In general, both orders of insects encode grps and pgrps . However, specific representatives of each class are restricted to lepidoptera (table 1). For example, the lepidopteran grp-2, which binds fungal cell wall -1,3 glucans and lta, is absent from diptera . Such derived grp and pgrps may contribute to lepidopteran - specific transduction of signals to downstream pathways . Other lepidoptera - specific prrs are hemolin and hemolymph proteinase-14 precursor (prohp14) (table 1). Like iml c - type lectins, hemolin is an lps- and lta - binding prr with roles in mediating cellular responses and as an opsonin to enhance phagocytosis . Hp14 has been shown to detect and bind a broad range of mamps, and may coordinate with grp1 or grp2 to activate propo [60, 106]. The potential role of the prrs discussed above in mediating the expression of amp genes remains to be determined, and further study of the lepidoptera - specific immune surveillance proteins and divergent activities of conserved prrs likely will yield novel avenues for pest - control . D. melanogaster has both mamp - dependent and mamp - independent routes to activate the toll pathway . In mamp - dependent toll induction, bacterial lys - pgn (typical of gram - positive bacteria) is detected by pgrp - sa or pgrp - sd (in the presence of gnbp-1), while yeast or fungal -1,3-glucan is detected by gnbp-3 [85, 108, 109] (figure 1). Mamp - independent stimuli are virulence determinants, such as proteases and chitinases, secreted by microbes and dubbed mamps and mamp - independent stimuli each trigger a distinct proteolytic cascade that both culminate in cleavage of the cytokine sptzle by the serine protease sptzle processing enzyme (spe). Interaction of active sptzle c - terminal domain (c-106) with the surface - localized toll receptor triggers an intracellular signal transduction terminating in induced expression of amps and cellular responses . Some of the basic components of the toll pathway are present in lepidoptera (figure 2). M. sexta hemocytes express an infection - induced toll - like receptor and the genome of b. mori encodes 14 genes predicted to encode toll - like receptors as well as homologs of each of the intracellular components of toll - dependent signaling [85, 114, 115]. Both m. sexta and b. mori encode homologs of the d. melanogaster toll - activating cytokine sptzle (figure 2). Also, for both m. sexta and b. mori there is experimental evidence linking the toll pathway with amp induction [116118]. In m. sexta, toll pathway results in expression of several antimicrobial peptides, including attacin-1, cecropin-6, moricin and lysozyme . In addition, the transcript level of hemolin, a pattern recognition protein exclusive to lepidopterans (table 1), is induced by injection of activated sptzle - c108 into larvae . Despite the conservation of certain aspects of the toll pathway, the extracellular cascades that lead to sptzle activation may have diverged between d. melanogaster and the two lepidoptera (figure 1). For example, in contrast to what is known in d. melanogaster, the m. sexta toll pathway is activated by gram - negative - associated mamps [115, 118]. Also, the genome of b. mori lacks 1:1 orthologs of grass, spirit and persephone, the d. melanogaster serine proteases responsible for mamp / prr - dependent and mamp - independent cleavage of spe (figure 1). Progress has been made in identifying a m. sexta proteolytic cascade that results in processing pro - sptzle into its active c - terminal domain (c-108). The direct cleavage is mediated by hemolymph proteinase (hp) 8 [11, 13], a homolog of d. melanogaster spe, in turn, hp8 is processed into its active form by hp6 . Hp6 is most similar to d. melanogaster persephone protease, which activates spe in response to mamp - independent stimuli [110, 113]. This hemolymph proteinase is activated in response to gram - positive or gram - negative bacteria and in response to -1,3-glucan . However, the prrs and proteolytic cascade that transduce mamp signals to amp induction are unknown (figure 1). The findings reviewed above demonstrate that while the overall architecture of the toll pathway is conserved among insects, the specific identities of proteolytic cascade members are distinct and many gaps remain in our understanding of toll activation in lepidoptera . Filling these gaps should reveal potential lineage - specific molecules that can serve as targets to hinder the activation of the toll pathway in agricultural pests . In d. melanogaster the imd pathway also contributes to amp gene induction and is triggered by direct interaction of dap - pgn, a mamp typical of gram - negative bacteria, with the transmembrane receptor pgrp - lc [82, 83, 125, 126]. For example, pgrp - le can act as an intracellular receptor for monomeric pgn and its truncated form can enhance pgrp - lc - mediated recognition . Dap - pgn / pgrp - lc interaction activates intracellular imd, which then recruits fas - associated death domain (fadd) and death - related ced-3/nedd2-like protein (dredd) to form a complex [129, 130]. Current evidence supports the idea that dredd, a caspase - like molecule, cleaves the nf-b transcription factor relish . Imd also appears to activate a phosphorelay: the transforming growth factor- (tgf)-activated kinase 1 (tak1) phosphorylates the ikb kinase (ikk) relish cleavage into its activated amino - terminal transcriptional regulator domain allows its translocation into the nucleus, where it activates amp gene expression . The translocation of relish into the nucleus is regulated by two recently discovered components of this pathway: inhibitor of apoptosis 2 (iap2) and transforming growth factor - activated kinase 1 (tak1)-binding protein 2 (tab2) [132, 133]. Both, iap2 and tab2 act upstream of relish and downstream of imd, while iap2 functions downstream of tak1 [132, 134]. Of particular importance to amp gene expression is iap2, the knockdown of which hampers the sustained expression of amp genes . While iap2 and tab2 are necessary for imd signal transduction, the gene product of pirk, a recently characterized gene, interacts directly with imd and pgrp - lc . Pirk overexpression analyses revealed that it acts as a negative regulator by reducing the expression of the amp genes attacin b, cecropin b, and diptericin b, which are all under the control of the imd pathway . Most of the information available about the imd pathway in lepidoptera comes from bioinformatics; orthologs of all intracellular components of the imd pathway have been found in b. mori [54, 137] and m. sexta . However, few experiments have been done to characterize the molecular mechanisms leading to activation of the imd pathway in these insects . In m. sexta several genes of the imd pathway, including those encoding imd, fadd, tak1, dredd and relish are up regulated in fat body of immune challenged 5th instar larvae and in the midgut of b. mori during the wandering stage . Genes encoding lysozyme, moricin and defensin amps also were up regulated in the midgut of b. mori in the wandering stage, consistent with the possibility that amp induction is imd - mediated . Finally, in the lepidopteran beet armyworm spodoptera exigua, rnai - mediated knockdown of relish expression resulted in loss of cecropin induction upon fungal infection, further strengthening the idea that the imd pathway may contribute to amp gene expression in lepidoptera, though perhaps it is triggered by distinct signals . Further study is needed to elucidate imd - mediated amp induction in lepidoptera and to reveal any differences there are in this pathway between diptera and lepidoptera . In d. melanogaster, transcription of amp - encoding genes is activated by the nf-b family transcription factors in response to infection [6569] with distinct nf-b family transcription factors responding to the toll and immune deficiency (imd) signal transduction pathways . In response to toll pathway activation, the nf-b inhibitor cactus is phosphorylated and degraded allowing its targets, the nf-b factors dif and dorsal, to be translocated to the nucleus . Imd pathway activity culminates in the nf-b factor relish being activated by a stimulus - induced proteolytic cleavage . In the case of dif and dorsal, gram - positive bacterial and fungal infections primarily serve as the stimuli that induce degradation of cactus through the toll signaling pathway . In general, gram - negative bacterial infections of d. melanogaster stimulate the proteolytic cleavage of relish through the imd pathway . Once in the nucleus, these transcription factors drive the transcription of immune effectors, including amp genes whose promoters contain nf-b binding sites [70, 72]. Overall, nf-b proteins and their dna - binding specificities are conserved among organisms, including those lepidoptera studied to date [73, 74]. However, the nfb - binding regions for inhibitor of b (ib) proteins (e.g., cactus) are not conserved, suggesting diversification and co - adaptation between ib and nfb pairs . Also, recent work indicates that nf-b nuclear co - regulators may contribute to species - specific regulation of amp gene expression . Therefore, modulation of inhibitors and nuclear - co - regulators of nf-b - dependent transcription may be one avenue by which target - specific immune suppression could be achieved . In d. melanogaster, nf-b - dependent amp induction through the toll and imd pathways is activated by detection of microbial components via different pattern recognition receptors (prrs). Prrs are soluble or membrane - bound proteins that bind to specific microbe associated molecular patterns (mamps) such as lipopolysaccharide (lps), lipoteichoic acid (lta), peptidoglycan (pgn) or -1,3-glucan that are released from or are found on the cell surfaces of bacteria or fungi . Upon interaction with mamps, prrs can directly agglutinate pathogens or trigger proteolytic signaling cascades and cytokine release, which in turn lead to the activation of downstream cellular and humoral pathways, including pro - po activation and amp gene expression [16, 65, 76]. Pgn recognition proteins (pgrps) and -1,3-glucanase - related proteins (grps) were discovered in the lepidopteran silkworm (b. mori) by assaying for plasma components that activate the propo cascade . Pgrps were subsequently shown to be conserved across mammals and insects, and in d. melanogaster their role in the induction of amp gene expression through toll and imd pathways in response to pgn has been well documented [7983]. Similarly, grps have been shown to induce amp expression through toll pathway in response to fungal infections [79, 84]. In contrast, there is a dearth of literature linking specific pgrps or grps to amp induction in lepidoptera . Such a link is possible, since pgn and -1,3-glucan can activate amp gene expression in m. sexta and b. mori [8590] and multiple infection - induced pgrp- and grp - encoding genes have been identified in lepidoptera [32, 38, 54, 55, 9194]. However, there are numerous hints that lepidoptera and diptera may have evolved divergent mechanisms of linking pathogen detection to conserved toll and imd signal transduction cascades . First, a genome comparison between b. mori and d. melanogaster failed to identify 1:1 pgrp orthologs . Similarly, b. mori gram - negative binding protein (gnbp) and m. sexta microbe binding protein (mbp), members of the -1,3-glucanase - related protein superfamily [76, 95], appear to be distantly related to d. melanogaster gnbps, suggesting divergence of this group of proteins . M. sexta mbp expression is strongly up - regulated in fat body after immune challenge and shows specific binding to lta, lps, dap - pgn . Also, in contrast to the situation in d. melanogaster, highly purified lps and lta are inducers of amp gene expression in lepidoptera, though not as potently as crude lps (with contaminating pgn) or purified pgn [85, 90, 96, 97]. This raises the possibility that different mamps or combinations of mamps are most efficacious in eliciting amp gene expression in lepidoptera relative to diptera . Also, since purified lps can elicit amp expression in lepidoptera but not d. melanogaster, lepidoptera have either a distinct repertoire of prrs responsible for lps - dependent triggering of imd or toll pathways, or an as - yet undiscovered pathway that links lps to amp induction . Testing these ideas awaits the identification of the suite of prrs and signal transduction pathways responsible for transducing lps, lta, pgn, or combinatorial microbial signals to amp gene expression . One class of lepidopteran prr that may mediate infection - dependent induction of amps is the c - type lectins (ctls), ca - dependent, secreted proteins that have carbohydrate - binding capabilities . Similar to some ctls of d. melanogaster, several ctls of m. sexta and b. mori [54, 99] are reported to mediate induction of cellular responses and the propo cascade . Although the nomenclature quickly becomes confusing, ctls include lipopolysaccharide - binding protein (lbp). M. sexta iml-1 binds to gram - positive and gram - negative bacteria as well as yeast, iml-2 shows specific binding to lps, iml-3 and iml-4 show specific binding to lps and lta, and iml-3 can also bind laminarin, a -1,3-glucan [103, 104]. Diversity in ctl carbohydrate - binding specificities may result in lineage - specific pathogen recognition - signal transduction connections . Of particular relevance to the theme of this review are prrs present in lepidoptera but not diptera (table 1). In general, both orders of insects encode grps and pgrps . However, specific representatives of each class are restricted to lepidoptera (table 1). For example, the lepidopteran grp-2, which binds fungal cell wall -1,3 glucans and lta, is absent from diptera . Such derived grp and pgrps may contribute to lepidopteran - specific transduction of signals to downstream pathways . Other lepidoptera - specific prrs are hemolin and hemolymph proteinase-14 precursor (prohp14) (table 1). Like iml c - type lectins, hemolin is an lps- and lta - binding prr with roles in mediating cellular responses and as an opsonin to enhance phagocytosis . Hp14 has been shown to detect and bind a broad range of mamps, and may coordinate with grp1 or grp2 to activate propo [60, 106]. The potential role of the prrs discussed above in mediating the expression of amp genes remains to be determined, and further study of the lepidoptera - specific immune surveillance proteins and divergent activities of conserved prrs likely will yield novel avenues for pest - control . D. melanogaster has both mamp - dependent and mamp - independent routes to activate the toll pathway . In mamp - dependent toll induction, bacterial lys - pgn (typical of gram - positive bacteria) is detected by pgrp - sa or pgrp - sd (in the presence of gnbp-1), while yeast or fungal -1,3-glucan is detected by gnbp-3 [85, 108, 109] (figure 1). Mamp - independent stimuli are virulence determinants, such as proteases and chitinases, secreted by microbes and dubbed mamps and mamp - independent stimuli each trigger a distinct proteolytic cascade that both culminate in cleavage of the cytokine sptzle by the serine protease sptzle processing enzyme (spe). Interaction of active sptzle c - terminal domain (c-106) with the surface - localized toll receptor triggers an intracellular signal transduction terminating in induced expression of amps and cellular responses . Some of the basic components of the toll pathway are present in lepidoptera (figure 2). M. sexta hemocytes express an infection - induced toll - like receptor and the genome of b. mori encodes 14 genes predicted to encode toll - like receptors as well as homologs of each of the intracellular components of toll - dependent signaling [85, 114, 115]. Both m. sexta and b. mori encode homologs of the d. melanogaster toll - activating cytokine sptzle (figure 2). Also, for both m. sexta and b. mori there is experimental evidence linking the toll pathway with amp induction [116118]. In m. sexta, toll pathway results in expression of several antimicrobial peptides, including attacin-1, cecropin-6, moricin and lysozyme . In addition, the transcript level of hemolin, a pattern recognition protein exclusive to lepidopterans (table 1), is induced by injection of activated sptzle - c108 into larvae . Despite the conservation of certain aspects of the toll pathway, the extracellular cascades that lead to sptzle activation may have diverged between d. melanogaster and the two lepidoptera (figure 1). For example, in contrast to what is known in d. melanogaster, the m. sexta toll pathway is activated by gram - negative - associated mamps [115, 118]. Also, the genome of b. mori lacks 1:1 orthologs of grass, spirit and persephone, the d. melanogaster serine proteases responsible for mamp / prr - dependent and mamp - independent cleavage of spe (figure 1). Progress has been made in identifying a m. sexta proteolytic cascade that results in processing pro - sptzle into its active c - terminal domain (c-108). The direct cleavage is mediated by hemolymph proteinase (hp) 8 [11, 13], a homolog of d. melanogaster spe, in turn, hp8 is processed into its active form by hp6 . Hp6 is most similar to d. melanogaster persephone protease, which activates spe in response to mamp - independent stimuli [110, 113]. This hemolymph proteinase is activated in response to gram - positive or gram - negative bacteria and in response to -1,3-glucan . However, the prrs and proteolytic cascade that transduce mamp signals to amp induction are unknown (figure 1). The findings reviewed above demonstrate that while the overall architecture of the toll pathway is conserved among insects, the specific identities of proteolytic cascade members are distinct and many gaps remain in our understanding of toll activation in lepidoptera . Filling these gaps should reveal potential lineage - specific molecules that can serve as targets to hinder the activation of the toll pathway in agricultural pests . In d. melanogaster the imd pathway also contributes to amp gene induction and is triggered by direct interaction of dap - pgn, a mamp typical of gram - negative bacteria, with the transmembrane receptor pgrp - lc [82, 83, 125, 126]. For example, pgrp - le can act as an intracellular receptor for monomeric pgn and its truncated form can enhance pgrp - lc - mediated recognition . Dap - pgn / pgrp - lc interaction activates intracellular imd, which then recruits fas - associated death domain (fadd) and death - related ced-3/nedd2-like protein (dredd) to form a complex [129, 130]. Current evidence supports the idea that dredd, a caspase - like molecule, cleaves the nf-b transcription factor relish . Imd also appears to activate a phosphorelay: the transforming growth factor- (tgf)-activated kinase 1 (tak1) phosphorylates the ikb kinase (ikk) relish cleavage into its activated amino - terminal transcriptional regulator domain allows its translocation into the nucleus, where it activates amp gene expression . The translocation of relish into the nucleus is regulated by two recently discovered components of this pathway: inhibitor of apoptosis 2 (iap2) and transforming growth factor - activated kinase 1 (tak1)-binding protein 2 (tab2) [132, 133]. Both, iap2 and tab2 act upstream of relish and downstream of imd, while iap2 functions downstream of tak1 [132, 134]. Of particular importance to amp gene expression is iap2, the knockdown of which hampers the sustained expression of amp genes . While iap2 and tab2 are necessary for imd signal transduction, the gene product of pirk, a recently characterized gene, interacts directly with imd and pgrp - lc . Pirk overexpression analyses revealed that it acts as a negative regulator by reducing the expression of the amp genes attacin b, cecropin b, and diptericin b, which are all under the control of the imd pathway . Most of the information available about the imd pathway in lepidoptera comes from bioinformatics; orthologs of all intracellular components of the imd pathway have been found in b. mori [54, 137] and m. sexta . However, few experiments have been done to characterize the molecular mechanisms leading to activation of the imd pathway in these insects . In m. sexta several genes of the imd pathway, including those encoding imd, fadd, tak1, dredd and relish are up regulated in fat body of immune challenged 5th instar larvae and in the midgut of b. mori during the wandering stage . Genes encoding lysozyme, moricin and defensin amps also were up regulated in the midgut of b. mori in the wandering stage, consistent with the possibility that amp induction is imd - mediated . Finally, in the lepidopteran beet armyworm spodoptera exigua, rnai - mediated knockdown of relish expression resulted in loss of cecropin induction upon fungal infection, further strengthening the idea that the imd pathway may contribute to amp gene expression in lepidoptera, though perhaps it is triggered by distinct signals . Further study is needed to elucidate imd - mediated amp induction in lepidoptera and to reveal any differences there are in this pathway between diptera and lepidoptera . Insecticides are necessary to guarantee effective insect pest management in agricultural settings . However, the cost and off - target effects of these insecticides directly and indirectly increase economic burden; the latter by affecting beneficial arthropods such as pollinators . The study of insect immunity can provide tools for the development of target - specific cost - effective approaches to control agricultural pests . Directed suppression of pest immune defenses is predicted to render them susceptible to environmental and applied biocontrol pathogens, as recently demonstrated in termites by bulmer and colleagues . The studies summarized above suggest that many aspects of insect immunity, including recognition factors and serine proteases, have diverged between d. melanogaster and lepidoptera . Continued comparative immunity of a broad array of species from diptera, lepidoptera, and other insect orders will reveal possible candidate immunity factors for target - specific approaches that will enable the effective control of insect pests . However, before such approaches can be realized, the details of lepidopteran immune signaling pathways must be elucidated . The relatively large sizes of last instar larvae of many lepidopteran species will facilitate biochemical approaches to such studies, while the establishment of immune - inducible lepidopteran cell lines such as the uga - cie1 cell line can enable the characterization of molecular mechanisms leading to imd pathway activation and its contribution to amp gene expression . Finally, ongoing investigations into the immune - modulatory mechanisms of entomopathogens will help identify key steps in immunity that are susceptible to manipulation, contributing to the development of natural, cost - effective, non - toxic alternatives to chemical insecticides currently used for pest management.

Syncope is caused by transient diffuse cerebral hypoperfusion and is characterized by transient loss of consciousness with a rapid onset followed by spontaneous and complete recovery . Clinical features of syncope may include myoclonic jerks which are often multifocal and asynchronous, convulsions, and urinary incontinence, making it difficult to differentiate from epileptic seizure by clinical features alone . Significant fluctuations in cerebral perfusion pressure are prevented by autoregulation of cerebral circulation, but there may be conditions where such mechanism may not compensate adequately . Cough syncope, a rare form of syncope, may be a result of transient failure of the cerebral autoregulatory mechanism to cope with sudden decrease in cerebral blood flow . We present an unusual case of recurrent cough syncope, which was initially diagnosed and treated as seizures, in the context of a left - sided glomus jugulare tumor, a benign paraganglioma . A 43-year - old right - handed woman with history of glomus jugulare tumor in the left jugular fossa with intracranial extension into the posterior cranial fossa was transferred from another hospital for recurrent seizure - like spells . She had a 90% surgical resection of the tumor done in 2011 followed by radiation therapy in september 2012 . Her episodes occurred multiple times a day (7 per day on average) during wakeful state . They were triggered by coughing (usually a bout of cough) and were characterized by staring and unresponsiveness as well as stiffening of the body with mild shaking of both upper extremities . She was diagnosed with epileptic seizures but continued to have episodes during treatment with the antiepileptic drugs (aeds) phenytoin, levetiracetam, and lamotrigine . Escalation of aed therapy made her increasingly drowsy, and she was on all three aforementioned aeds at the time of presentation . Her physical examination was remarkable for excessive drowsiness, mild dysarthria, right sixth cranial nerve palsy, mild hypertonia with hyperreflexia in the lower extremities (left more than right), and bilateral (left more than right) ankle clonus . She had a lumbar puncture done at the outside hospital, and the opening cerebrospinal fluid (csf) pressure was reported to be 25 cm . Blood work was also unremarkable except for mild anemia (hemoglobin: 9.4 g / dl), mild hyponatremia (132 meq / l), and mild hypokalemia (3.1 meq / l). Antiepileptic drug levels were within therapeutic range (free phenytoin: 1.3 g / ml, levetiracetam: 5.9 g / ml, and lamotrigine: 2.3 g / ml). All started with a bout of cough when the patient was lying in bed (in supine or in lateral position) which was followed by brief (less than a minute) distal upper extremity tremor and subtle proximal upper extremity myoclonic jerks and prolonged unresponsiveness for up to 10 min . All of these episodes were associated with hypotension (7278/3147 mm of hg as revealed by continuous arterial pressure monitoring) and bradycardia (5459 bpm). The eeg during the spells was characterized by generalized synchronous and asynchronous high amplitude 1- to 2-hz delta activity which progressed to generalized attenuation and then transitioned to generalized delta activity again with recovery (fig . 1). A head ct showed recurrence of the glomus jugulare tumor and communicating hydrocephalus . An external ventricular drain (evd) after placement of the evd, her drowsiness gradually started to improve, and episodes decreased in frequency to one per day . 3 showed an enhancing t2 hyperintense left skull base mass in the region of the left jugular foramen with extension into the posterior cranial fossa and below the base of the skull . Brain imaging showed evidence of hydrocephalus that had increased compared with her previous brain imaging done 2 months back . Her mental status continued to improve, and she had only one mild episode triggered by cough during the next two days before her discharge . Repeat surgical resection of the tumor was recommended by the otolaryngology team, which the patient declined . Based on the clinical features and eeg findings, the episodes observed in our patient are most consistent with cough syncope . The mechanism underlying cough syncope is not definitively established, but it is postulated that coughing increases intrathoracic and intraabdominal pressures leading to a transient increase in icp . Increased icp, in turn, causes a decrease in cerebral perfusion pressure which, if it drops below a critical level, may result in global cerebral hypoperfusion leading to syncope . Transient cerebral circulatory arrest has been demonstrated by transcranial doppler measurements during cough syncope . Our patient also had a drop in blood pressure and heart rate but probably not sufficient to cause syncope by itself . Cough syncope has been associated with posterior fossa mass lesions or tonsillar herniation and with hydrocephalus . It may be speculated that bouts of cough caused transient herniation of cerebellar tonsils obstructing csf flow that further contributed to the increase in icp during coughing . Decrease in frequency of events following placement of evd to relieve icp lends support to this notion . Paragangliomas are rare tumors of extraadrenal chromaffin cell origin that most commonly occur in the head and neck region . Catecholamine - hypersecreting paraganglionomas are uncommon in the head and neck region, and most patients (95%) with hypersecreting paraganglionomas have hypertension . Hypotension accompanying syncope observed in our patient was not orthostasis - related (the patient was always supine during spells) and was most likely related to cough . Identified a subset of patients with cough syncope who lacked a blood pressure overshoot (expected response) after the relief of straining during valsalva maneuver . The authors postulate that cough syncope in these patients might be the result of delayed recovery from hypotension that follows a paroxysm of cough, and this was likely contributing to global cerebral hypoperfusion in our patient . This case highlights the fact that cough syncope, a rare form of syncope, may be associated with intracranial mass lesions that indirectly exaggerate the increase in icp in response to cough . Glomus caroticum tumor presenting as recurrent unexplained syncope and posterior fossa meningioma presenting as recurrent cough syncope have been described . Recurrent cough syncope should trigger search for factors, including brain tumors, with the potential to cause transient elevation in icp . This case also illustrates an important role for ceeg monitoring with video in distinguishing syncope from seizures in cough syncope cases.

World - wide, infertility affects 1015% of couples who are trying to conceive, and about 15% of these cases are caused by male factors, which affect 1 out of 20 men in the general population . Most cases of male infertility are idiopathic, apart from several etiologies, such as obstruction of deferent duct, varicocele, sexual dysfunction, and cryptorchidism . Although assisted reproductive technology (art) has helped many sterile couples to conceive, non - obstructive azoospermia (noa), which accounts for a considerable proportion of male infertility, has a dramatically lower rate of sperm retrieval and clinical pregnancy . The etiological mechanism of noa is unknown, but factors such as oxidative stress were considered to have effects on spermatogenesis, and some antioxidants have been effective in protecting spermatogenesis . Therefore, it is helpful to explore the underlying pathogenesis of noa in these patients . Micrornas are a class of small rnas that do not code amino acid sequences, but they play fundamental roles in regulating gene expression after transcription . Lian et al . Found 154 down - regulated mirnas and 19 up - regulated mirnas in testes of noa patients compared to fertile males, by using microarray technologies . Furthermore, some of the mirnas have been shown to affect the proliferation, apoptosis, and dna damage in germ cells [911]. Mir-210 is one of the 19 up - regulated mirnas in testes of noa patients, located within the genomic loci of transcript ak123483 . It can be induced by hypoxia, and plays an essential role in cell adaptation to hypoxia . Mir-210 also affects regulation of diverse physiological processes, such as angiogenesis, cell survival, proliferation, cell cycle arrest, protein modification, and dna damage repair . Although mir-210 has been shown to be involved in regulation of physiological processes in various diseases and to be an up - regulated mirna in testes of noa patients, it remains unknown how mir-210 affects spermatogenesis . Hence, the aim of this study was to investigate the underlying mechanisms by which mir-210 is involved in the pathogenesis of spermatogenesis . We enrolled 25 patients (aged 1841 years) with azoospermia (proven by 3 semen analyses from testicular biopsies from the first affiliated hospital of anhui medical university). Pathological examinations were performed on each testicular specimen . Combined with clinical features, 4 patients were diagnosed as having sertoli - cell - only syndrome (scos), 7 patients were diagnosed as having maturation arrest (ma), 8 patients were diagnosed as having hypospermatogenesis, and the other 6 patients were diagnosed as having obstructive azoospermia (oa). All patients provided informed consent before their participation in this study . Our local medical ethics committee approved this study before it began . To examine the location of insulin - like growth factor ii (igf2) in human testicular tissues, we performed immunohistochemistry staining to detect the igf2 expression . Tissues were cut into sections for immunoperoxidase staining after being treated with 4% pfa and paraffin wax . After the specific treatment with standard - procedure immunohistochemistry staining as described as lian et al ., sections were incubated with igf2 antibody (abcam) overnight at 4c and biotinylated secondary antibody (abcam) for 2 h at room temperature . To detect expression of mir-210, rnas were extracted from nt-2 cells or tissues and subjected to real - time pcr as described as lian et al . . Briefly, rna extraction was performed following a standard trizol protocol, real - time pcr was carried out with the abi step one system (applied biosystems), and the sybr premix ex taq ii kit (takara bio, inc .) Was used . Primers for q - rt pcr were as follows: forward primer: 5-caataactgtgcgtgtgacagc-3 reverse primer: 5-tatggttttgacgactgtgtgat-3 forward primer: 5-cagcacatatactaaaattggaacg-3 reverse primer: 5-acgaatttgcgtgtcatcc-3 western blot analysis was carried out to detect protein expression of igf2 in the human testicular tissues in the 3 groups and in nt2 cells . Anti - igf2 (abcam) was used for western blot analysis, and we used -actin as a loading control to detect expression of igf2 . We supplemented the medium with 10% fetal bovine serum (life technology inc . ), 1% antibiotics (100 units / ml penicillin, and 100 ug / ml streptomycin, life technology inc . ). Cells were incubated at 37c in a humidified incubator with 5% co2 . To transfect oligonucleotides and plasmids into nt-2 cells, lipofectamine rnaimax (invitrogen) and fugene hd (roche) all processes were performed in accordance with the protocols supplied by manufacturers . In this study, all the experiments were performed independently at least 3 times . We enrolled 25 patients (aged 1841 years) with azoospermia (proven by 3 semen analyses from testicular biopsies from the first affiliated hospital of anhui medical university). Pathological examinations were performed on each testicular specimen . Combined with clinical features, 4 patients were diagnosed as having sertoli - cell - only syndrome (scos), 7 patients were diagnosed as having maturation arrest (ma), 8 patients were diagnosed as having hypospermatogenesis, and the other 6 patients were diagnosed as having obstructive azoospermia (oa). All patients provided informed consent before their participation in this study . To examine the location of insulin - like growth factor ii (igf2) in human testicular tissues, we performed immunohistochemistry staining to detect the igf2 expression . Tissues were cut into sections for immunoperoxidase staining after being treated with 4% pfa and paraffin wax . After the specific treatment with standard - procedure immunohistochemistry staining as described as lian et al ., sections were incubated with igf2 antibody (abcam) overnight at 4c and biotinylated secondary antibody (abcam) for 2 h at room temperature . Rnas were extracted from nt-2 cells or tissues and subjected to real - time pcr as described as lian et al . . Briefly, rna extraction was performed following a standard trizol protocol, real - time pcr was carried out with the abi step one system (applied biosystems), and the sybr premix ex taq ii kit (takara bio, inc .) Was used . Primers for q - rt pcr were as follows: forward primer: 5-caataactgtgcgtgtgacagc-3 reverse primer: 5-tatggttttgacgactgtgtgat-3 forward primer: 5-cagcacatatactaaaattggaacg-3 reverse primer: 5-acgaatttgcgtgtcatcc-3 western blot analysis was carried out to detect protein expression of igf2 in the human testicular tissues in the 3 groups and in nt2 cells . Anti - igf2 (abcam) was used for western blot analysis, and we used -actin as a loading control to detect expression of igf2 . We supplemented the medium with 10% fetal bovine serum (life technology inc . ), 1% antibiotics (100 units / ml penicillin, and 100 ug / ml streptomycin, life technology inc . ). Cells were incubated at 37c in a humidified incubator with 5% co2 . To transfect oligonucleotides and plasmids into nt-2 cells, lipofectamine rnaimax (invitrogen) and fugene hd (roche) the igf2 gene is part of a cluster of imprinted genes expressing the single polypeptide as igf2, which is only produced from the paternal allele . The maternal allele is transcriptionally silent . To clarify the location of igf2 in human testicular tissues, we found igf2 located in spermatocytes in the testes of patients with oa (figure 1). Because igf2 is located in spermatocytes of the testis, we detected the expression of igf2 in cases with ma, hypospermatogenesis, and oa, but not in the scos patients . We found that igf2 was down - regulated in patients with ma and hypospermatogenesis compared to oa patients, which was considered as the control group with normal spermatogenesis, although without a significant difference (figures 2, 3), possibly because there were fewer samples and longer preservation times of some samples . Quantitative real - time pcr was performed to examined mir-210 expression in the testis of patients with ma, hypospermatogenesis, and oa . We found that mir-210 was significantly up - regulated in the testis of ma and hypospermatogenesis patients compared to oa patients (figure 4). However, due to errors in the rna extraction in the preliminary experiment, 3 testis samples (1 each) from ma, hypospermatogenesis, and oa patients were damaged and were not tested . In the targetscan database, because the 3utr of the igf2-mrna has a putative mir-210-binding site, igf2 was predicted to be a potential target of mir-210 . To identify whether the igf2 gene was targeted by mir-210 directly, renilla luciferase reporters, which include the wild - type full - length 3utr forms of mir-210 seeding sites, figure 5 shows that there was a 60% decrease in luciferase activity after cotransfection of the mir-210 mimic and the renilla luciferase reporters into nt2 cells, and inhibiting mir-210 expression increased activity of the reporter renilla luciferase . Expression of igf2 protein was also significantly lower in the nt2 cells transfected with mir-210 mimics than in control cells, and knockdown of mir-210 with mir-210 inhibitor increased protein expression of igf2 (figures 6). The igf2 gene is part of a cluster of imprinted genes expressing the single polypeptide as igf2, which is only produced from the paternal allele . The maternal allele is transcriptionally silent . To clarify the location of igf2 in human testicular tissues, we found igf2 located in spermatocytes in the testes of patients with oa (figure 1). Because igf2 is located in spermatocytes of the testis, we detected the expression of igf2 in cases with ma, hypospermatogenesis, and oa, but not in the scos patients . We found that igf2 was down - regulated in patients with ma and hypospermatogenesis compared to oa patients, which was considered as the control group with normal spermatogenesis, although without a significant difference (figures 2, 3), possibly because there were fewer samples and longer preservation times of some samples . Quantitative real - time pcr was performed to examined mir-210 expression in the testis of patients with ma, hypospermatogenesis, and oa . We found that mir-210 was significantly up - regulated in the testis of ma and hypospermatogenesis patients compared to oa patients (figure 4). However, due to errors in the rna extraction in the preliminary experiment, 3 testis samples (1 each) from ma, hypospermatogenesis, and oa patients were damaged and were not tested . In the targetscan database, because the 3utr of the igf2-mrna has a putative mir-210-binding site, igf2 was predicted to be a potential target of mir-210 . To identify whether the igf2 gene was targeted by mir-210 directly, renilla luciferase reporters, which include the wild - type full - length 3utr forms of mir-210 seeding sites, were used . Figure 5 shows that there was a 60% decrease in luciferase activity after cotransfection of the mir-210 mimic and the renilla luciferase reporters into nt2 cells, and inhibiting mir-210 expression increased activity of the reporter renilla luciferase . Expression of igf2 protein was also significantly lower in the nt2 cells transfected with mir-210 mimics than in control cells, and knockdown of mir-210 with mir-210 inhibitor increased protein expression of igf2 (figures 6). During recent decades several studies have focused on the effects of mirnas on spermatogenesis in male infertility [911,17]. However, it was not understood how mir-210, which is one of the up - regulated mirnas in testes of patients with noa, was involved in spermatogenesis in male infertility . The transformation of diploid spermatogonia into mature haploid cells in spermatogenesis is a complex biological process in the testes of males . The insulin / igf system takes part in the processes of cell proliferation, cell growth, differentiation, and survival, which affects nearly every organ in the body . Also, insulin / igf plays an important role in the proper function of the testis in males . Igf2 binds to igf1r and insr - a with a high affinity and binds to insr - a / igf1r, insr - b / igf1r, but with lower affinity . Found that in inactivated insr and igf1r, there was a 79% reduction in daily sperm production in adult mouse testes by a conditional ko approach . Taken together, the aforementioned data suggest that igf2 might be involved in the process of spermatogenesis . To examine the specific mechanism by which mir-210 is associated with the process of spermatogenesis, quantitative real - time pcr was performed to detect mir-210 expression . We found that mir-210 was significantly up - regulated in the testes of subjects with ma and hypospermatogenesis patients compared to oa . These results agree with findings of lian et al . Using microarray technologies performed in noa and normal controls . Several studies have suggested that this mirna could be mediated by hypoxia and participate in various types of regulation of angiogenesis, cell survival, proliferation, cell cycle arrest, and protein modification [1214]. Furthermore, some researchers even found that mir-210 might be considered as one of the indicated markers in some diseases, such as clear cell renal cell carcinoma and acute myeloid leukemia . In the present study we found that igf2 was targeted by mir-210 directly in the in vitro experiment in nt2 cells, and mir-210 might be associated with spermatogenesis by targeting igf2 in male infertility . Firstly, as some errors occurred in the rna extraction in the preliminary experiment, mir-210 of 3 testes samples were damaged and not detected in the subsequent quantitative real - time pcr experiment, which might have affected our results . Secondly, we did not investigate the functions of mir-210 and igf2 in vitro or in vivo, and we plan to do this in future research . We demonstrated that mir-210 might be associated with spermatogenesis by targeting igf2 in male infertility . Future mechanistic studies on the role of mir-210/igf2 in the process of spermatogenesis in male infertility will provide new insights into the diagnosis and management of male infertility.

Midwife - led primary delivery care for low - risk pregnant women during labor has been reported to have various advantages, such as increased odds of high maternal satisfaction and a decrease of unnecessary medical interventions [18]. Although the maternity care system for low - risk pregnant women peculiar to one country cannot easily be compared with those in other countries, consumer demands for the humanization of obstetric care have arisen in various countries [18]. To date, we have found no evidence that midwife - led primary obstetric care is unsafe for low - risk pregnant women in comparison with obstetric care with the favorable cooperation of obstetricians and midwives in japan [912]. In addition, about 85% of low - risk pregnant women request that they give birth while receiving midwife - led primary delivery care . Therefore, safe midwife - led delivery care with the backup of obstetricians may also be required for low - risk pregnant women in japan . If complications occur or threaten to occur during the primary midwife - led delivery care, the midwives have to refer the woman to obstetricians at the same or a neighboring hospital or private obstetric clinic as soon as possible . This is because, in deliveries managed by independent midwives in japan, many intervention measures, such as oxytocin infusion, epidural anesthesia, episiotomy, suture, and instrumental delivery, are not available based on japanese legal restrictions . In our institute, one of the main tokyo city perinatal centers, there are 3 japanese systems of midwife - led delivery care, as follows: (1) those intending to give birth at home managed by midwives who do not belong to our hospital, (2) those planning to give birth on futons (i.e., japanese - style bedding) in japanese tatami mat delivery rooms in our hospital managed by the same midwives who do not belong to our hospital, and (3) those planning to give birth in japanese tatami mat delivery rooms managed by midwives who belong to our hospital . The objective of this study was to describe trends in transfers and perinatal outcomes among labors using these 3 japanese systems of midwife - led primary delivery care . The protocol for this analysis was approved by the ethics committee of the japanese red cross katsushika maternity hospital . In addition, informed consent for analysis from a retrospective database was obtained from each subject during their hospital visit . In our hospital, pregnant women who are initially considered low - risk at 3436 weeks of gestation can choose freely between the 3 systems of midwife - led care and obstetric shared care . In the midwife - led care units, midwives can practice autonomously and are fully accountable for their own practice, unsupervised by obstetricians . Factors used to exclude women from the low - risk group comprise the following [912]: (1) medical history: pregnancy - induced hypertension, chronic hypertension, diabetes mellitus, renal disease, idiopathic thrombocytopenia, and other systemic illnesses; (2) gynecological history: history of infertility therapies of in vitro fertilization, congenital uterine anomalies, uterine myomatosis, and adnexal anomaly; (3) obstetric history: narrowing of the pelvic outlet, cephalopelvic disproportion, previous cesarean section, previous anal sphincter injury, previous postpartum hemorrhage 1,000 ml with blood transfusion, previous manual removal of placenta, previous gestational diabetes, and history of severe preeclampsia; (4) complications during the present pregnancy: multiple pregnancy, nonvertex presentation, obesity (maternal body mass index before pregnancy 25 and/or during the third trimester 28), anemia (hemoglobin <9.0 g / dl), epilepsy with treatment, polyhydramnios, oligohydramnios, low - set placenta, placenta previa, fetal growth restriction, heavy for date fetus, gestational diabetes, and pregnancy - induced hypertension; when risk factors are present, those women are managed by obstetricians and midwives; (5) complications during labor: intrauterine infection, thick meconium staining, prolongation of labor such as active - phase dilation <1 cm / hour and duration of second stage of labor 2 hours, prolonged rupture of membranes (24 hours), uterine inertia, arrest of labor, and fetal heart rate abnormality such as a nonreassuring fetal status . When these factors are present, the women are transferred to be managed mainly by obstetricians (obstetric shared care) in a standard western - style delivery room or surgery room in our hospital . A retrospective study was performed to examine trends in transfers and perinatal outcomes among labors that started using the 3 systems of midwife - led primary delivery care . In this study, student's t - test was used for continuous variables and the test for categorical variables . Odds ratios (ors) and 95% confidence intervals (cis) were also calculated . Differences with p between 2009 and 2012, a total of 678 low - risk women were placed in the 3 forms of midwife - led primary delivery care at the onset of labor pains and/or rupture of membranes at 3741 weeks of gestation . Of these, 123 (18%) intended to give birth at home, 88 (13%) planned to give birth in the japanese tatami mat rooms in our hospital managed by midwives who do not belong to our hospital, and 467 (59%) planned to give birth managed by the midwives belonging to our hospital . Table 1 shows the clinical descriptions of the 678 pregnant women initially considered as low - risk for receiving our midwife - led primary delivery care systems . There were no significant differences in the maternal age or parity among the 3 groups . Table 2 shows the rate of transfers in the 3 groups of the midwife - led primary delivery care systems . The total rate of transfers in the system run by the midwives belonging to our hospital (56%) was higher than in the other 2 systems run by the independent midwives (31% in planned home birth: or 1.87, 95% ci 1.23.0, p <0.01; 38% in planned hospital birth: or 2.51, 95% ci 1.73.8, p <0.01). In addition, the timing of transfers in the system run by the midwives belonging to our hospital (before the second stage of labor: 52%) was earlier than those in the other 2 systems (21% in the planned home birth: or 4.12, 95% ci 2.66.6, p <0.01; 20% in planned hospital birth: or 4.29, 95% ci 2.57.4, p <0.01). However, if classified into nulliparous and parous women, there were no significant differences in the rate of transfers among the 3 groups, as shown in table 1 . In addition, among the 3 groups there were no significant differences in the rate of the main 2 indications for transfer: fetal heart rate abnormality and failure to progress . The main indications for transfer after delivery were maternal postpartum hemorrhage and neonatal respiratory distress associated with asphyxia . Table 3 shows the obstetric and neonatal outcomes in the pregnant women initially considered as low - risk for receiving our midwife - led primary delivery care systems . There were no significant differences in these outcomes among the 3 groups our obstetric care system involves the division of women in labor into low- and high - risk groups [912]. The women who are initially considered low - risk can choose freely between midwife - led care and obstetric shared care . If complications occur or risk factors arise during labor in the primary midwife - led care, they are transferred to obstetric shared care . This may be the first report concerning the differences in the timing of transfers from midwife - led care to obstetric shared care among the 3 systems of midwife - led primary delivery care in japan . In this study, there was no evidence that the primary midwife - led care is unsafe for low - risk pregnant women in any of these 3 midwife - led delivery care systems . However, there were no significant differences in the timing of referrals from midwife - led care to obstetric shared care between the system led by midwives who belong to our hospital (hospital midwifery system) and the systems led by the midwives who do not belong to our hospital . In the hospital midwifery system, the timing of transfers seemed to be the earliest due to the ease of transfer within the same hospital and administrator setting . On the other hand, the rate of transfers after delivery with the other 2 systems was higher than that in the hospital midwifery care . During the period, the main indications for transfers were maternal postpartum hemorrhage and/or neonatal respiratory distress associated with asphyxia . Fortunately, the difference was not associated with adverse obstetric or neonatal outcomes; however, unfortunately, they led to early mother - to - child separation, especially in cases of planned home birth because healthy puerperal women or newborns cannot be transferred from home to hospital according to japanese law . Although home birth might be very comfortable, those involved must be prepared for mother - to - child separation in cases of referrals after delivery . The major limitations of this study were the small sample size and lack of long - term follow - up of mothers and children to consider the potential of the findings based on our own context . There were no cases of fetal / neonatal death under the midwife - led delivery care . The most evaluated outcome under midwife - led delivery was the satisfaction of pregnant women with the development of mother - child relationships after delivery . In addition, there might be some bias related to the backgrounds in the selection of the systems because this was not a randomized trial study . Therefore, a further large prospective study with long - term follow - up may be needed . There were no significant differences in perinatal outcomes among the 3 systems; however, there were some differences in the status of the transfers to the obstetric shared care.

Diabetes decreases the overall life expectancy and cause a heavy burden on public health (1). Moreover, the asia - pacific region is considered to be on the verge of an emerging diabetes epidemic (2). The development of type 2 diabetes is affected by genetic and environmental determinants (3). Recently, one study investigated whether common variants of functional and positional candidate genes, including adrb3, pparg, enpp1 and capn10, were determinants of type 2 diabetes (4). Enpp1, also called k121q, has a glutamine substitution for lysine at codon 121 (5). Type 2 diabetes is characterized by insulin resistance (6), and enpp1 plays an important role in insulin resistance (7, 8). Enpp1 interacts with -subunit of the insulin receptor to interrupt signaling (9). In previous studies, the k121q polymorphism of the human pc-1 gene was strongly associated with insulin resistance (1, 3, 7, 10 - 12). However, there was no association between insulin resistance and the k121q variant (13, 14). In addition, there were discrepancies for the impact of enpp1 polymorphism on obesity between ethnic groups (5, 10, 13, 15 - 19). Obesity increases the concentration of insulin in plasma and is the major contributor of insulin resistance (20). Obesity appears to be an effect modifier of type 2 diabetes in d1057 carriers (21). The association of obesity with the genetic variant of the insulin receptor substrate was identified in other studies (21, 22). In the chinese han population, the pc-1 q121 allele was associated with insulin resistance . In women, carriers of the q allele had an increased risk for obesity development (3). In caucasians and african - americans, 121q carriers had an association with increased body mass index (bmi) (23), and the three - allele risk type haploid qdeltg with the q allele increased the risk for obesity (24) however, in the danish population, there were no differences in the distribution of frequencies of dominant types (kk wild type and kq / qq variant type) and alleles (19). The complexity of type 2 diabetes is related to factors such as genetic heterogeneity, interactions between genes, and the modulating role played by the environment (4). In spite of these limitations, studies of type 2 diabetes and genetic factors of obesity can predict the risks for development of both type 2 diabetes and obesity in order to assist primary prevention, and korea is an appropriate country for such studies because of the homogeneity of racial composition and lifestyle (25). Therefore, the aim of this study was to analyze the presence of the enpp1 polymorphism, not studied yet in korean population, to identify the association between genotypes and allele with type 2 diabetes and obesity . This company is an electric power company located at kori, yonggwang, ulchin, wolsung, and seoul in korea . There were 195 male workers (age 48.26.7 yr, bmi 24.672.64 kg / m) who were diagnosed as diabetics during medical examinations conducted from march 2004 to october . The 1,750 male workers (age 45.27.7 yr, bmi 24.772.64 kg / m) in the control group were selected randomly . Subjects were included if they met one of the criteria below and their onset age was older than 20 yr old to exclude type 1 diabetes: 1) blood - sugar level before a meal exceeded 126 mg / dl twice or more; 2) blood - sugar level before a meal exceeded 126 mg / dl once or more and blood - sugar level two hours after a meal exceeded 200 mg / dl; 3) those who reported that he had a history of diabetes in the questionnaire and being taken oral hypoglycemic agents . The workers included in the obesity group were those whose bmi was 25 kg / m or more . After collecting a blood sample from vein in fasting (8 hr) status, we measured the blood - sugar level, insulin, and lipid profile . We also measured height and weight to calculate bmi . If fasting glucose levels were greater than 126 mg / dl, we checked the 2 hr post - prandial plasma glucose level at each site within 1 month . Height and weight were measured by autoanalyzer (health guard, fanics, seoul, korea). The fasting blood level was analyzed by glucose - oxidase assay using an autochemistry analyzer . To determine the lipid profile, total cholesterol was analyzed by enzyme assay using cholesterol oxidase (cod), high density lipoprotein (hdl) cholesterol by glycerol phosphate oxidase assay, low density lipoprotein (ldl) cholesterol by direct surfactant assay . We carried out this study under the approval by the ethnics committee of the asan medical center, and obtained written consent from all subjects, providing subjects with sufficient explanation to obtain informed consent . We extracted genomic dna from buffy coats using the generall blood sv kit (general biosystem, seoul, korea) and following the instructions suggested by the manufacturer . The method of genotyping used to identify the k121q polymorphism in enpp1 exon 4 was polymerase chain reaction - restriction fragment length polymorphism (pcr - rflp), using dna treated with a restriction enzyme after pcr, based on the paper reported by abate et al . We carried out student's t - test to analyze the effects of genotypes on biochemical parameters using genotypes as factors . The hardy - weinberg equilibrium was computed based on the goodness - of - fit test . We also investigated the differences in frequencies of genotypes between type 2 diabetic and normal groups, and between obesity and normal groups by fisher's exact test . The spss 12.0 (for window) statistical software package was used for statistical analysis . This company is an electric power company located at kori, yonggwang, ulchin, wolsung, and seoul in korea . There were 195 male workers (age 48.26.7 yr, bmi 24.672.64 kg / m) who were diagnosed as diabetics during medical examinations conducted from march 2004 to october . The 1,750 male workers (age 45.27.7 yr, bmi 24.772.64 kg / m) in the control group were selected randomly . Subjects were included if they met one of the criteria below and their onset age was older than 20 yr old to exclude type 1 diabetes: 1) blood - sugar level before a meal exceeded 126 mg / dl twice or more; 2) blood - sugar level before a meal exceeded 126 mg / dl once or more and blood - sugar level two hours after a meal exceeded 200 mg / dl; 3) those who reported that he had a history of diabetes in the questionnaire and being taken oral hypoglycemic agents . The workers included in the obesity group were those whose bmi was 25 kg / m or more . After collecting a blood sample from vein in fasting (8 hr) status, we measured the blood - sugar level, insulin, and lipid profile . We also measured height and weight to calculate bmi . If fasting glucose levels were greater than 126 mg / dl, we checked the 2 hr post - prandial plasma glucose level at each site within 1 month . Height and weight were measured by autoanalyzer (health guard, fanics, seoul, korea). The fasting blood level was analyzed by glucose - oxidase assay using an autochemistry analyzer . To determine the lipid profile, total cholesterol was analyzed by enzyme assay using cholesterol oxidase (cod), high density lipoprotein (hdl) cholesterol by glycerol phosphate oxidase assay, low density lipoprotein (ldl) cholesterol by direct surfactant assay . We carried out this study under the approval by the ethnics committee of the asan medical center, and obtained written consent from all subjects, providing subjects with sufficient explanation to obtain informed consent . We extracted genomic dna from buffy coats using the generall blood sv kit (general biosystem, seoul, korea) and following the instructions suggested by the manufacturer . The method of genotyping used to identify the k121q polymorphism in enpp1 exon 4 was polymerase chain reaction - restriction fragment length polymorphism (pcr - rflp), using dna treated with a restriction enzyme after pcr, based on the paper reported by abate et al . We carried out student's t - test to analyze the effects of genotypes on biochemical parameters using genotypes as factors . The hardy - weinberg equilibrium was computed based on the goodness - of - fit test . We also investigated the differences in frequencies of genotypes between type 2 diabetic and normal groups, and between obesity and normal groups by fisher's exact test . The spss 12.0 (for window) statistical software package was used for statistical analysis . The frequencies of the kk type, kq type, and qq type in enpp1 k121q were 82.1%, 17% and 0.9%, respectively . The homozygous type of q carrier (qq type) was added to the heterozygous type (kq type) because the frequency of qq type was very low (0.9%). We investigated the differences in age, blood pressure, bmi, and results of clinical examinations according to genotypes of enpp1 k121q between the type 2 diabetic and non - diabetic groups (table 1). When type 2 diabetics and non - diabetics were pooled (n=1,945), there were no significant differences in bmi, systolic pressure, diastolic pressure, glucose value in fasting status, total cholesterol, ldl cholesterol, hdl cholesterol, triglyceride, c - reactive protein, and homa - ir between the kk type and kq+qq type . In addition, there were no significant differences in the same characteristics above between type 2 diabetic group and non - diabetic groups (table1), and between the obese group and non - obese groups (table2). The frequencies of genotypes were in accordance with the hardy - weinberg equilibrium (p=0.85). The odds ratio of having a kq+qq genotype was 0.85 for diabetics versus non - diabetics . The odds ratio of having a q allele was 0.91 for diabetics versus non - diabetics . However, there was no significant difference in the genotypic and allelic distribution between type 2 diabetics and non - diabetics (table3). The odds of a kq+qq genotype were 0.93 for obese versus non - obese subjects . Furthermore, the odds of having a q allele were 0.96 for obese versus non - obese subjects . However, there was no significant difference in the genotypic and allelic distribution between obese and non - obese (table4). The frequency of the kk type in enpp1 k121q genotypes was 82.1%, that of kq+qq type was 17.9% and that of q allele was 9.4% . There was no statistically significant difference in the distribution among these genotypes and alleles (p=0.81, p=0.89, respectively), although the 121q carrier and q allele in obese and/or diabetics seemed to differ slightly from those in the non - obese non - diabetics (reference group). In determining the prevalence of the q allele carriers (kq and qq subjects) and q allele, there were no significant differences in the genotypic and allelic distribution with the respect to any phenotypes (data not shown). After adjusting for the effects of obesity, the probability of type 2 diabetes in kq+qq type was 0.858 (data not shown) and that in q allele was 1.095, showing no significant difference (table 5). Moreover, after adjusting for the effects of type 2 diabetes, the probability of developing obesity in the kq+qq type was 0.936 (data not shown) and that in the q allele was 1.038, showing no significant difference (table 6). The results of studies of the association between enpp1 k121q variants and both type 2 diabetes and obesity in several races are disparate . We carried out this study to investigate the association of k121q variants with type 2 diabetes and obesity in korean male workers . Insulin resistance is a major component of the pathogenesis of type 2 diabetes (27), and insulin receptor kinase activity is impaired in muscle and other insulin - sensitive tissue of many type 2 diabetic patients (28), and then a potential inhibitor of the insulin receptor tyrosine kinase is identified as the plasma - cell membrane differentiation antigen-1 (pc-1) (29). Therefore, it is significant to analyze enpp1 (pc-1) polymorphism . In the previous studies of the association between type 2 diabetes and polymorphism in enpp1, the k121q missense mutation increased the odds ratio (or) for type 2 diabetes in dominican (10), south asian, caucasian (16), finnish (18), and french populations (24). Moreover, according to a meta - analysis of the association between enpp1 k121q variant and type 2 diabetes, the odds ratios were 1.30 (95% confidence interval [ci], 1.13 - 1.50) (16) and 1.17 (95% ci, 1.10 - 1.25) (19), showing significant association . In this study, 121q was not associated with type 2 diabetes, showing consistent results with those in the japanese population (5), danish caucasians (13), oji - cree population (17), finnish population (18), and danish white subjects (19). Most type 2 diabetes in koreans is characterized by non - obesity, thus the enpp1 k121q mutant relevant to insulin resistance possibly could be a candidate gene that is not appropriate to explain susceptibility to type 2 diabetes . This can be a possible explanation for the lack of association between enpp1 121q carrier and type 2 diabetes in this study . Obesity is a main risk factor for the development of type 2 diabetes (20) and there is linear association between obesity and type 2 diabetes (3). In previous studies of the association between obesity and polymorphism in enpp1, 121q carriers and/or q allele were associated with obesity in the chinese han population (bmi of obesity group 27 kg / m) (3), caucasians (bmi of obesity group> 90th percentile), african - american adults (bmi of obesity group> 80th percentile) (23), french population (bmi of obesity group 95th percentile) (24), and dominican population (bmi of obesity group 30 kg / m) (10). However, in this study (bmi of obesity group 25 kg / m), there was no difference in distribution between obesity and 121q carriers and presence of the q allele . This result was consistent with those from a study of 7,333 danes (19) and a spanish population (14) in which the bmi of the obesity group was 25 kg / m or higher . On the other hand, in matsuoka's study, the percentage of subjects whose bmi was 30 kg / m or higher was too low (2.5%) to investigate the effect of k121q genotype on obesity . The results of the present study showed that the frequencies of the q allele was 8.7% in the type 2 diabetic group and 9.2% in the obesity group, which was lower than those in finnish and swedish populations (12.9 - 15.1%) (11), danish caucasians (14 - 16%) (13), south asians in chennai (14%), caucasians in dallas (16%), south asians in dallas (19%) (16), the dominican population (54.2%) (10) and, black children (77%) (30). The frequencies of the q allele investigated in this study might have a smaller statistical power to explain any association with either type 2 diabetes or obesity with 121q carriers (kq+qq) and/or the q allele . In conclusion, the present study suggests that the enpp1 k121q polymorphism was not associated with type 2 diabetes and obesity . The results of negative associations in this study might be attributable to the low prevalence of obesity, relatively younger age, and low frequencies of the 121q carriers . Large and prospective studies are needed to confirm this preliminary observation in the korean population.

Lipid apheresis provides a safe and effective means of treating patients with severe hyperlipidemia . It functions by first separating plasma from blood cells with a cell separator and then using either the adsorption of apolipoprotein b by affinity columns containing anti - apolipoprotein b antibodies or dextran sulphate, or their precipitation at low ph by heparin . Lipid apheresis allows patients to attain lower levels of low - density lipoprotein (ldl), which are usually not attainable with traditional drug therapy alone, while leaving high - density lipoprotein (hdl) levels generally unaffected . When used in conjunction with statins and other lipid - lowering drugs, lipid apheresis may also induce the regression of coronary atherosclerotic plaque in familial hypercholesterolemia (fh) patients . Fh is a group of autosomal dominant genetic defects resulting in elevated serum (ldl) cholesterol levels . In the heterozygous state, fh is a relatively common but serious genetic disorder, with an incidence of about 1 in 500 persons in the general population . Fh has been associated with an increased risk for atherosclerosis, premature coronary heart disease, and heart failure [3, 4]. Fh is caused by a mutation affecting apolipoprotein b, proprotein convertase subtilisin kexin type 9 (pcsk9; an enzyme involved in ldl receptor degradation), or, most commonly, the ldl receptor gene, resulting in defective ldl receptors and/or a diminished number of ldl receptors [7, 8]. These mutations cause ldl to be catabolized at a slower rate and thus accumulate in the circulation . Currently, fh is treated using a variety of cholesterol - lowering drugs, most notably statins or hmg - coa reductase inhibitors . For many patients, however, statins are not a viable treatment option, because of either intolerance or ineffectiveness . Lipid apheresis is an alternative form of treatment for these fh patients as well as those who have persistently elevated ldl levels despite treatment . Because apheresis is performed at only a few highly specialized centers in relatively low volume, there is very little literature discussing the effectiveness of lipid apheresis on the reduction of lipid profiles and the prevention of future cardiac events . This study, therefore, reports the experience in a single metropolitan center of treating patients with hyperlipidemia with lipid apheresis . Retrospective chart reviews were performed and questionnaire surveys were given to active lipid apheresis patients at the minneapolis heart institute (mhi) at abbott northwestern hospital (anw), minneapolis, minnesota . Mhi and anw are divisions of allina health, a large healthcare provider in minnesota and western wisconsin . Patients were identified through an electronic health record (ehr) screen of ambulatory patients representing all patients seen at all allina health metro area and regional locations between 2009 and 2012 (epic systems, verona wi). Of these patients, criteria to qualify for apheresis were based on the united states food and drug administration (fda) approval recommendations . Currently, the fda supports ldl apheresis for patients who, after six months, do not have an adequate response to diet therapy and maximum drug therapy, due to either ineffectiveness or intolerance, and meet the following criteria: functional homozygotes with an ldl cholesterol> 500 mg / dl without cad, functional heterozygotes with ldl cholesterol> 300 mg / dl without cad, functional heterozygotes with ldl cholesterol> 200 mg / dl with documented coronary heart disease . Functional homozygotes with an ldl cholesterol> 500 mg / dl without cad, functional heterozygotes with ldl cholesterol> 300 mg / dl without cad, functional heterozygotes with ldl cholesterol> 200 mg / dl with documented coronary heart disease . The date of birth, gender, date of apheresis initiation, lipid disorder diagnosis, apheresis frequency, and family history of cardiac events were recorded . Patients were noted as having fh if the active problem list contained a diagnosis of fh . To determine which patients had fh, we used the national lipid association (nla) criteria for an 80% probable fh diagnosis, using the highest ldl recorded in the patient chart as follows: age <20 and ldl> 190 mg / dl, age 2029 and ldl> 220 mg / dl, and age 30 and ldl> 250 mg / dl . Potential homozygous fh (hofh) patients were defined as having an untreated ldl> 500 mg / dl or a treated (on statin) ldl over> 300 mg / dl, in addition to clinical evidence of xanthomas before age of 10 years or having two parents with heart disease or high lipids . Identifiable secondary causes for marked hyperlipidemia were excluded from the analysis by examining the ehr chart of each potential homozygote . Current cholesterol lowering medications were also recorded, focusing on the use of statins, colesevelam (welchol), ezetimibe (zetia), niacin, and aspirin . A significant cardiovascular event was defined as a myocardial infarction (mi), a percutaneous transluminal coronary angioplasty (ptca) or stenting procedure, or a coronary artery bypass graft (cabg) using ehr documented icd-9 criteria . Cardiac events were separated by their occurrence before or after the patient began apheresis, and the total number of events was recorded for each group . Multiple cardiac events occurring at the same hospitalization, such as mi followed by ptca, were counted as a single event for cardiac event rate calculation . Pre- and postapheresis cardiac event rates were calculated by adding the total number of cardiac events and dividing by the total person years during each time period . The preapheresis time period describes the time from the first documented ehr visit to the date of apheresis initiation . The postapheresis time period describes the time from the date of apheresis initiation to the study date . Unverifiable events noted in the ehr but occurring prior to the first documented ehr visit were noted but excluded from the cardiac event rate calculation . Mean acute ldl reductions were calculated by averaging all recorded ldl values prior to and immediately after the treatment sessions . Mean acute total cholesterol, hdl cholesterol, and triglyceride reductions were calculated by using lipid profile from the most recent treatment session . Ldl apheresis was performed at abbott northwestern hospital using the kaneka liposorber la-15 system (kaneka medical products). The system consists of the kaneka ma-03 machine, the integrated sulflux kp-05 plasma separator, which consists of porous hollow fibers, to separate the plasma from the whole blood, and two disposable liposorber la-15 adsorption columns to adsorb apolipoprotein b - containing lipoproteins from patient plasma . Patients confirmed information in their ehr such as risk factors, answered questions relating to their awareness of fh and if their family had been previously tested for it, provided their level of satisfaction with their apheresis program, and indicated their interest in learning more about alternative treatments . The data from the questionnaires was cross - referenced with the data from the patient charts to ensure accuracy . Descriptive statistics are displayed as means and sds for continuous variables; number and percentage with characteristic are given for categorical variables . Categorical variables were analyzed using pearson's chi - square or fisher's exact tests . Continuous variables were analyzed using student's t - test . A value of p <0.05 was considered significant, and p values are two - sided where possible . All statistical calculations and plots were done with stata 11.2 (college station, tx). Institutional review board approval was obtained for data collection, follow - up, and data analysis . Of these, 8 (72.7%) were male, 10 (90.9%) were caucasian, 1 (9.1%) was african american, 10 (90.9%) carried the diagnosis of fh, with 2 (18.2%) patients identified as probable homozygotes, and 1 (9.1%) was diagnosed as having familial combined hyperlipidemia . The average age of patients was 65.6 9.3 years, and patients had been on apheresis for an average of 6.2 7.0 years . Four (36.4%) patients were currently on statins while the other 7 (63.6%) had a history of statin intolerance . Five of 11 (45.5%) patients were on a nonstatin cholesterol lowering medications, including 1 (9.1%) on colesevelam (welchol), 3 (27.3%) on ezetimibe (zetia), and 1 (9.1%) on niacin . Maximum ldl levels ranged from 211 to 448 mg / dl with a mean (sd) value of 298 80.7 mg / dl in the study group . Since our ehr was implemented in 2005, it is possible we may be underestimating the highest lifetime ldl for each patient . Of the 11 participants, 9 completed the questionnaire in its entirety; 1 patient provided answers to all questions but did not disclose risk factors and 1 patient did not complete the questionnaire . All of the patients indicated that they were aware that they likely had fh and 7 patients indicated that their immediate family had been tested for fh . The patients self - reported a total of 44 cardiac events before apheresis and 8 cardiac events after apheresis . Of the 10 patients that completed the questionnaire, 4 patients were currently on statins while the other 6 were statin intolerant . Eight patients (72.7%) had a cardiac event documented by ehr, with 43 cardiac events occurring overall (table 4). Self - reported events which were unable to be verified via the ehr were excluded from the cardiac event rate analysis . Thirty - four cardiac events were documented before apheresis in 8 patients compared with 9 events in 5 patients after apheresis . After excluding cardiac events that were unverifiable, 14 cardiac events were documented in the preapheresis time period and 7 were documented in the postapheresis time period . The cardiac event rates were calculated to be 0.23 (0.13, 0.39) events per person year in the preapheresis group and 0.10 (0.041, 0.21) events per person year in the postapheresis group (p = 0.064). Patients were observed for an average of 7.6 5.9 years before apheresis and 6.2 4.7 years after apheresis, with 60.6 total patient years before apheresis and 67.8 patient years after apheresis . This study was conducted to gain more information on lipid apheresis and evaluate the effectiveness in lowering lipid values . In addition, through chart review and patient survey, we attempted to gain a greater understanding of this patient population in terms of traditional risk factors, family awareness and screening, statin and other cholesterol medication uses, desire for additional treatment options, and ultimately cardiac events . Our study shows that apheresis markedly lowers total cholesterol, ldl cholesterol, triglycerides and, to a much lesser degree, hdl cholesterol . There was a small, but statistically significant, reduction in hdl values after apheresis . Many of these patients were statin intolerant and some had been using nonstatin cholesterol medications . Importantly, 10/11 (90.9%) participants indicated a desire to learn more about other potential treatment options, indicating that this population may indeed experience fatigue of this procedure . Although taken from a small study population, our data suggests a reduction in cardiac event rate after apheresis . While not statistically significant, our data shows a strong trend towards event rate reduction . This statistical insignificance is likely explained by the study's small sample size . With a larger population it is also important to note that the risk for cardiac events increases with age . Ldl apheresis has been shown to improve endothelium dependent vasodilation [11, 12], microvascular flow, and myocardial perfusion . Some studies [2, 15, 16] have also shown a significant reduction in angiographic cad, but others have not . These studies have been small, primarily nonrandomized trials . The ldl - apheresis atherosclerosis regression study (laars) looked at the change in plaque characteristics of patients undergoing apheresis compared to drug therapy over a period of two years . In that period, 7 out of 21 patients on apheresis had a cardiac event compared with 3 out of 21 on medication only . While this study found that apheresis arrested the progression of atherosclerosis the fh regression study found that ldl apheresis combined with simvastatin was more effective than colestipol plus simvastatin in reducing ldl cholesterol and lipoprotein (a) but was less effective at influencing coronary atherosclerosis . Another study found that, out of 18 patients, 3 had myocardial infarctions, 1 underwent a cabg, and 12 needed coronary angioplasties within two years of beginning a combination therapy of apheresis, statins, and probucol . Before beginning the combination therapy, 11 had experienced a mi, 5 had undergone a cabg, and 13 had undergone an angioplasty . The heparin - induced extracorporeal ldl precipitation (help) study found that help is suitable for reducing ldl concentrations and may work to reduce the burden of atherosclerosis, as there were no myocardial infarctions and a low coronary intervention rate in patients who began apheresis . Due to the expensive nature of apheresis, a randomized, controlled clinical trial is needed to truly gauge the effectiveness of apheresis in reducing the occurrence of cardiac events . If apheresis is not deemed effective or is minimally effective, as some of these studies suggest, other types of treatment, such as lomitapide, mipomersen, or pcsk9 inhibitors, should be pursued . While satisfaction was generally high in our survey, patients specifically cited that this satisfaction was based on the results of apheresis and not on the process itself . Many patients complained about the invasive nature of apheresis, citing bruises from the procedure and the inconvenience of reporting for treatment every two weeks . Additionally, almost all patients were interested in learning more about alternative treatments, suggesting that they would prefer an alternative treatment which could match the results provided by apheresis . This study had several limitations . Since lipid apheresis is an advanced treatment for an uncommon genetic disease, the limited number of patients available to participate in the study led to a small sample size . The event rate reduction was not statistically significant but showed a strong trend toward cardiac event rate reduction before and after apheresis . By defining the observational period initial time point as the first documented ehr visit, we excluded 20 events before apheresis and 2 events after apheresis from the cardiac event rate calculation . The lipid - lowering effects of apheresis are best expressed as reductions in interval means . Although lipid apheresis was performed every two weeks, ldl values were not measured ever two weeks due to clinical practices . This inconsistency in measurement intervals prevents the use of more advanced measures to accurately track the effect apheresis has on ldl measurements . Finally, this study focused on active apheresis patients and therefore did not include patients who had stopped apheresis or were deceased . Lipid apheresis can reliably reduce ldl, non - hdl cholesterol, triglyceride, and total cholesterol levels in fh patients . Our data suggest that lipid apheresis shows a strong, but not statistically significant, trend towards the reduction of cardiac events . Apheresis is a viable treatment for fh patients, especially those that are statin - intolerant, due to its lipid lowering nature and its apparent reduction of cardiac events . However, there is a need for alternative treatments which are less invasive and provide easier patient access.

Agenesis of the inferior vena cava (ivc) as a cause of recurrent deep vein thrombosis (dvt) is uncommon . A 33-year - old male with no family history of thrombophilia, who had experienced multiple recurrent episodes of dvt over a 15-year period of unknown cause, was admitted into our hospital because of cellulitis in the right leg . Congenital absence of the ivc could be a rare risk factor for idiopathic dvt, especially in young individuals . Venous thromboembolism (vte), which includes deep vein thrombosis (dvt) and pulmonary embolism, has an incidence of 1 to 3 per 1000 individuals per year in western populations.1 congenital anomalies of the inferior vena cava (ivc) are uncommon, and have been associated with the development of venous thrombosis of the lower limbs.2 congenital anomalies of the ivc has been reported as a risk factor for dvt, especially in individuals <30 years old, and a concomitant thrombophilic disorder has been found in such individuals.3 we report a case of recurrent dvt in a 33-year - old man with agenesis of the ivc . The patient had experienced recurring episodes of idiopathic dvt in the right leg for 15 years . A 33-year - old man was admitted to the internal medicine department, holy family hospital, nazareth, israel, because of cellulitis in the right leg . One week prior to his admission, he complained about pain and increased local heat in the left ankle and thumb of the right leg . The patient had no history of previous trauma, surgery, insect bites, dysuria, or joint symptoms, and no family history of thrombophilia . He reported that he had (a) rheumatic fever without any complications when he was 19 years old, which was treated with penicillin, (b) been hospitalized when he was 23 years old because of infected skin ulcers on the right calf, for which he was treated by parenteral antibiotics, and (c) recurrent episodes of idiopathic dvt for the last 15 years . He also reported that he had not been treated with warfarin, but he had been on prophylactic enoxaparin therapy for dvt some years ago which has since been stopped and that he had been recently treated with allopurinol and colchicine for a presumed diagnosis of gout . He had been investigated several times for a primary hypercoagulability state, and the results were negative . On examination, the most outstanding clinical findings were swelling of ankles, mild edema, redness, and increased temperature of the right ankle and calf with trophic skin changes (skin discoloration with ulcers), and superficial varicose veins in the lower abdomen (figure 1). The clinical laboratory findings (erythrocyte sedimentation rate, leukocyte and platelet counts, and plasma hemoglobulin, plasma protein c, plasma protein s, fibrinogen, and antithrombin iii levels), the results of the kidney and liver function tests, and resistance to activated protein c were all normal . Polymorphisms of the genes that encode for methylenetetrahydrofolate reductase were not detected, and the factor v leiden and prothrombin mutations g20210a were absent . The results of the clinical immunological studies for complement c3 and c4 and rheumatoid factor were negative, and no circulating titers for antinuclear antibody, antineutrophil cytoplasmic antibody, and cardiolipin antibody were found . Cultures from the infected skin ulcers of the right leg were positive for methicillin - resistant staphylococcus aureus (mrsa). Ultrasound imaging of the leg veins showed a previous dvt in the right common femoral vein, and dilated superficial inguinal veins . Computer tomography with contrast of the abdomen showed agenesis of the infrarenal segment of the ivc (figure 2) with dilated azygos and hemiazygos veins (figure 3). There were also varicose veins in the abdominal wall and right groin, which were associated with dilated superficial and collateral veins (figure 4). Transthoracic echocardiography of the patient s heart revealed mild atrial enlargement and good systolic function of the left ventricle, and no pathological valvular flows . The patient was diagnosed as having agenesis of the infrarenal segment of the ivc and dvt of the right leg without concomitant risk factors for vte . Since we attributed the agenesis of the ivc to be the underlying cause of the recurrent episodes of the dvts, the patient was started on anticoagulant therapy (subcutaneous enoxaparin 160 mg / day) for dvt, antibiotic therapy (intravenous vancomycin 1.5 g / day for mrsa skin infection), and referred to a vascular surgeon specialist but the patient refused . At follow - up in the internal medicine clinic, the most outstanding clinical findings were swelling of left ankle, redness, with trophic skin changes, and a mild improvement of the skin ulcers . Despite several phone calls for follow - up the normal ivc is composed of 4 segments: hepatic, suprarenal, renal, and infrarenal . Since many transformations can occur during the formation of the ivc such anomalies occur in 0.3% of otherwise healthy individuals, and in 0.6% to 2% of patients with other cardiovascular anomalies.4 ruggeri et al reported 10 years ago 4 cases of congenital absence of the ivc in 75 young patients with idiopathic dvt over a 5-year period, and estimated that 5% of young patients with dvt had an anomaly of the ivc.5 venous thrombosis is caused by the presence of isolated or combined risk factors . Almost 150 years ago, the nineteenth century pathologist rudolf virchow described 3 critically important causes of venous thrombosis: venous damage, coagulation defect(s), and venous stasis.6 individuals with a congenital anomaly of the ivc are typically asymptomatic, and the anomaly is usually detected incidentally during radiological or abdominal procedures . Congenital absence of the ivc is infrequently associated with thromboembolic events.5 patients who suffer from congenital anomalies of the ivc usually develop a compensatory circulation through the azygos veins or collateral abdominal veins in order to keep the venous return near normal levels.7 most reported cases of congenital anomalies of ivc cases have been linked to thrombophilia disorders.3,5,7 however, the true prevalence of thrombophilia in congenital anomalies of the ivc is unknown because the screening for thrombophilia in patients with an ivc anomaly was usually incomplete.3 anticoagulants, but not thrombolytic therapy, are usually prescribed for venous thrombosis, but the duration of the anticoagulant therapy is not well established . Hence, anticoagulant therapy for an indefinite duration will probably be prescribed, unless vascular reconstructive surgery is done on the anomalous ivc . Such surgery has been rarely reported, and its long - term outcome is undetermined.8 congenital anomalies of the ivc may cause recurrent dvt, especially in young individuals.

An exponential rise in alzheimer's disease (ad) prevalence rates is predicted to parallel the aging of baby boomers creating a potentially unsustainable economic burden to the healthcare system . Delaying the onset or progression of ad, even modestly, by earlier pharmacological intervention could substantially reduce the economic and psychosocial impact of the illness [1, 2]. Unfortunately, many ad patients remain undiagnosed or go undetected until the later stages of disease . Insights into the underlying pathological mechanisms involving beta - amyloid plaque deposition within the brain have led to the development of a host of antiamyloid agents that are in various stages of clinical investigation . There is now a scientific consensus that the pathological events in ad initiate decades before clinical symptoms become apparent, and if disease modification is realized in the coming decades, the need for improved methods of early detection prior to the overt clinical signs will be accentuated . Traditionally, neuropsychological measures, particularly those that tap cognitive abilities subsumed by the hippocampal formation such as episodic memory, have shown usefulness in identifying cognitively normal elders who subsequently develop ad [4, 5]. Decrements in semantic memory and concept formation have been shown to occur nearly a decade before the development of ad . Performance on visual - spatial and verbal memory measures in midlife have also been shown to predict later memory loss . However, individuals with very high premorbid intellectual abilities experiencing incipient cognitive decline may go undetected, and false positives are possible in individuals with a low level of intellectual abilities . Also appropriate interpretation of extensive neuropsychological testing requires a high degree of expertise and training, which limits its use in routine clinical settings . The advancement of molecular imaging tracers that bind to amyloid, such as pittsburgh compound b (pib) or longer - lived probes (e.g., fddnp), offers a non - invasive in vivo method to detect and quantify brain amyloid deposition [8, 9]. However, this approach for presymptomatic detection is economically impractical for routine use given the current costs and restrictions on medically necessary use . Similarly, biomarkers including a142 and phosphorylated tau (also implicated in ad pathology) in cerebral spinal fluid (csf) can predict subsequent cognitive decline [10, 11], but lumbar puncture carries risks and is inconvenient for wide - scale use in cognitively impaired elderly subjects . Blood - based biomarkers have more practical applicability for routine use and are likely to be more cost effective than both csf and imaging procedures . Consequently, measurement of a140 and a142 in blood is increasingly being explored and shows potential in identifying individuals at the preclinical stage of ad [1214]. It has been reported that csf a levels are subject to high diurnal fluctuations with extremely high variability reported over 12 hours . Over days and weeks, furthermore, serum contains more a than plasma, possibly due to the release of bound a during the clotting process . Hence, serum a appears suitable for use in predicting mci / ad and optimal sensitivity, and specificity is probably achievable if combined with current diagnostic procedures, such as brief neuropsychological testing . In this study, we examined the usefulness of brief neuropsychological tests in combination with blood a140 and a142 as a predictive test for detecting mci / ad in at - risk older adults at a pre - symptomatic stage . Such an approach will be more practical for clinical use and be germane in designing large - scale prevention trials . Participants included a subset of subjects enrolled in the alzheimer's disease anti - inflammatory prevention trial (adapt). Adapt was a randomized, placebo - controlled, multicenter primary prevention trial sponsored by the national institute on aging . Subjects were randomly assigned to one of three groups: celecoxib (200 mg b.i.d . ), naproxen sodium (220 mg b.i.d . ), or placebo . Full details of data collection, measurements, and study procedures are available at http://www.jhucct.com/adapt/manall43.pdf and described elsewhere . The inclusion criteria for adapt subjects were age of 70 or older at enrollment, a self - reported family history of ad - like dementia, and normal cognitive performance on a brief battery of neuropsychological tests . Recruitment for adapt began in 2002, and the study was completed in 2007 . In 2005, the roskamp institute initiated a proteomic ancillary study (f. crawford, pi) involving blood draw from these subjects . The inclusion criteria for this ancillary study stipulated that each subject was an active adapt participant and had met all the adapt inclusion and exclusion criteria . A separate consent was also obtained from each subject who participated in the ancillary study . Two hundred and fifteen subjects from the roskamp adapt cohort enrolled in the proteomic ancillary study . At the time of blood draw, subjects maintained cognitively normal status as determined by their performance on an annual cognitive assessment battery . Blood was collected during the semi - annual followup visits, and the cognitive assessments were performed at the baseline visit and at the annual visits . The time from baseline cognitive testing to the diagnosis of mci / ad was 4.06 years (1.3 sd). Timeframe from baseline cognitive testing to blood draw was 2.25 years (0.71 sd) and from blood draw to diagnosis was 1.79 years (1.2 sd). The cognitive measures completed at baseline and annual followup included the modified mini - mental state examination (3ms); the hopkins verbal learning test - revised (hvlt - r); digit span (forward and backward) from the wechsler adult intelligence scale - revised (wais - r); a generative verbal fluency test (supermarket items); the narratives from the rivermead behavioral memory test (rbmt); the brief visuospatial memory test - revised (bvmt - r). The mini - mental state examination (mmse) was extracted from 3ms . Alternate forms were utilized annually for the hvlt - r, rbmt, and bvmt - r on each subsequent annual visit . Subjects also completed the 30-item geriatric depression scale and a self - rating scale of memory functions . Collateral respondents completed the dementia severity rating scale (dsrs). Due to significant intercorrelations between these tests, analyses described below are limited to those baseline cognitive tests that were sensitive to early changes (i.e., verbal learning and memory) associated with mci / ad or tests that were similar to those previously shown to be associated with a levels . Normative data from the cache county study was used to develop the standardized cut - off scores utilized in adapt . Individuals who scored below the cut scores on annual cognitive assessments underwent further dementia workup including physical and neurological examinations, laboratory studies (i.e., cbc, chemistry count, sedimentation rate, vitamin b12 and folic acid levels, thyroid test, and syphilis serological test), and neuroimaging (i.e., mri or ct), as applicable . A more comprehensive neuropsychological assessment was also administered by a neuropsychologist as part of the dementia work - up . This battery of tests consisted of the expanded consortium to establish a registry for alzheimer's disease (cerad) battery; logical memory i and ii of the wechsler memory scale - revised; benton visual retention test (benton); a generative fluency test (animals); control oral word association test (cowat; cfl); the trail making test; symbol digit modalities test (smdt); shipley vocabulary . Following completion of all components of the dementia work - up, a consensus team determined cognitive status using published diagnostic criteria . The diagnosis of ad was made using nincds - adrda and amnestic mild cognitive impairment (mci) using petersen criteria . All mci patients were considered to be amnestic mci, as they only had memory impairment, but maintained normal activities of daily living and overall had a well - preserved cognition in other cognitive domains . Ample evidence indicates that amnestic mci patients may be in a transitional stage between normal aging and ad with 85% of these subjects converting to ad over a 7-year period . Additional evidence comes from an imaging study which demonstrated that the pattern of brain atrophy in amnestic mci patients is typical of that observed in ad patients . It is then reasonable to combine these diagnoses in a single category, thus allowing a large enough numbers to supply statistical power . Of the 215 subjects who gave blood for the ancillary study, two developed non - ad dementia, and of the remaining subject pool of 208 used in these analyses, 28 subjects met criteria for either ad (n = 10) or mci (n = 18) in the two years following blood draw . The serum a content was determined, as per manufacturer's instructions, using the elisa kits for human a140 and a142 and the inter - assay cv, and the intraassay cv was reported to be 10% (invitrogen, calif). Dna was extracted from whole blood for apoe genotyping using pure gene kits (gentra systems, calif), and apoe genotyping was performed using previously established methods, as described elsewhere . Apoe genotypes were unavailable for 4 individuals, but these were included in the analyses . The data set was range checked, and prior to analyses, the dependent and independent variables were examined for missing data, outliers, and violations of the normalcy assumption . Differences among groups on demographic variables, neuropsychological variables, and serum a140 levels were examined using either the student's t - test or analyses, depending on the type of variable measurement . Time - updated cox regression modeling was used to test whether neuropsychological test scores, a, or a combination of both can predict conversion to mci / ad in individuals who were cognitively normal at baseline . Potential confounding variables shown to impact risk for cognitive decline included age, education, gender, apoe status, serum creatinine, triglycerides, presence of apoe 4 allele, and history of vascular disease as determined by treatment with statins or antihypertensive medication which were entered as covariates . The latter variables, coded dichotomously, have been previously shown to impact a levels . Because previous analyses revealed a nonsignificant increase of ad risk with naproxen in this cohort, we also controlled for this effect . Logistic regression modeling was employed to construct receiver operator curves (roc) to examine the predictive performance of neuropsychological measures from the baseline visit and serum a levels in diagnoses of mci / ad . Roc curve comparisons were based on area under the curve (auc), se, and the associated 95% confidence interval (ci). We subsequently calculated sensitivity of the various models using the predicted probability of each subject by logistic regression modeling with specificity of at least eighty percent . Post hoc power calculations using the g - power software for multivariate regression analyses utilized here suggest a power of nearly 100% at the alpha value 0.05 for the current sample size, total number of predictors, and the observed effect size . The mean age and education of the sample was 76.7 (sd = 3.9) and 14.6 (sd = 2.8) years, respectively . The majority of the sample was caucasian (98.1%), and 51.9% were male . Despite the cohort's self - report of enriched family history, less than one - third of the total sample (31.7%) carried at least one apoe 4 allele, a frequency similar to the general population . Comparisons on variables between subjects who remained cognitively normal and those who declined over the short follow - up period are reported in table 1 . Although all subjects at enrollment performed within the normal limits based on the established cut - off scores, those that ultimately declined had generally poorer scores on the 3ms, mmse, and all memory measures . The two groups were also significantly different on serum a142 levels and a142/a140 ratios prior to diagnoses of mci / ad . Only 23% of the cognitively normal individuals had serum a142 in the lowest quartile compared to the nearly 50% of the diagnostic group (44% of mci subjects and 50% of ad subjects). Time - dependent cox regression analyses were performed to examine the relationship between these cognitive tests and a on the prediction of subsequent conversion to mci / ad . All neuropsychological analyses were adjusted for age, gender, and education, but no adjustment for the study medications was required as these were baseline scores . Cox regression analyses show that the model using neuropsychological tests predicted mci / ad (2 log - likelihood = 206.51, = 52.11, df = 8, p <.001). Significant individual neuropsychological measures were 3ms (= 0.25 0.06, wald = 17.78, p <.001); generative verbal fluency (= 0.12 0.04, wald = 8.09, p <.004); hvlt - r scores (= 0.24 0.11, wald = 4.58 p <.032). Cox regression analysis showed that a142 measured in the lowest two quartiles compared to the highest quartile was a significant individual predictor of conversion to mci / ad in this model (2 log - likelihood = 197.47, = 38.41, df = 15, p <.001). The regression analysis utilizing the a142/a140 ratio found similarly significant results (2 log - likelihood = 204.69, = 36.10, df = 14, p <.001) with the lowest ratios being most predictive of subsequent conversion to mci / ad . The final full model, adjusting for confound and the study medications, included hvlt - r, fluency, 3ms, a142 levels, and a142 quartiles (2 log - likelihood = 166.25, = 74.55, df = 18, p <.001) with fluency, 3ms, and a142 in the lowest two quartiles as significant individual predictors of mci / ad in the model . Similar results were observed when a140 levels and a142 quartiles were substituted in this model with a142/a140 ratios (2 log - likelihood = 168.49, = 72.90, df = 17, p <.001). Baseline values for the 3ms, hvlt - r, and generative verbal fluency scores were subtracted from those obtained at the 12-month repeat testing to determine if changes in these measures differ by a142 and a142/a140 ratios . In unadjusted analyses, among subjects who converted to mci / ad, the greatest decline for hvlt - r was observed among individuals with the lowest quartile of a142 (1.17, 2.33 sd) and a142/a140 ratios (0.75, 2.63 sd) where individuals in the highest quartile of a142 (1.33, 1.86 sd) and a142/a140 ratios improved by nearly one point (0.6 1.82 sd). However, these differences were not statistically significant (p>.05). For the 3ms scores, among subjects who converted to mci / ad, those with a142 in the lowest quartile declined (1.83 1.28 sd) as compared to the highest quartile (4.83 1.35 sd), and this difference was statistically significant (f = 3.42, p = .033). For mci / ad subjects with the lowest quartile of the a142/a140 ratios, the 3ms values remained ultimately unchanged (0.16 1.20 sd), while the scores improved among those with the highest quartile of the a142/a140 ratios (4.33 1.20 sd), and these differences were also statistically significant (f = 3.10, p = .046). For generative verbal fluency test, a decline was noted in both the lowest quartile (4.17 1.40 sd) and the highest quartile (1.17 2.13 sd) of a142, and these differences were marginally significant (f = 2.63, p = .073). For a142/a140 ratios, a similar pattern was observed, but this difference was not statistically significant . Among individuals who remained cognitively normal, while a similar pattern was observed, those with lowest quartile of a142 and a142/a140 ratios had a larger decline than those with the highest quartile for each hvlt - r (0.28 0.27 sd versus . 0.14 0.33 sd, respectively .) And 3ms (1.02 0.51 sd versus 0.39 0.44 sd). However, due to the small magnitude of the change in these scores, these differences were not statistically significant . No such change was observed for the generative verbal fluency test (data not shown). Examination of sensitivity and specificity using roc analysis revealed the auc for neuropsychological testing with age, education, and gender as covariates was 0.83 (95% ci [0.750.91], p <.001). For a142 (adjusted for presence of apoe 4 allele, vascular risk factors, and associated medications), the auc was 0.79 (95% ci [0.700.88], p <.001). When neuropsychological testing (3ms, hvlt - r, and generative verbal fluency) and a142 were combined, the auc was increased to 0.91 (95% ci [0.860.95], p <.001). For the adjusted (as above) a142/a140 ratios alone, .001), and when combined with the neuropsychological measures, auc was 0.91 (95%ci [0.870.96], p <.001). Optimal sensitivities with specificity of at least 80% predicted probabilities are shown in table 2 . The highest sensitivity and specificity was achieved using a combination of cognitive scores and a142/a140 ratio, but this finding was driven by a142 . The pathogenesis of ad is initiated before the clinical symptoms of cognitive impairment and functional decline become apparent in its victims . A simple and pragmatic method for identifying older adults at an increased risk for mci / ad who may benefit from targeted prevention is therefore of importance in reducing the burden of ad . The combination of brief neuropsychological tests along with blood - based biomarkers of ad represents a reasonable approach with a potential for wide - scale use . Our findings here provide support for this notion and demonstrate that early prediction of risk for developing mci / ad may be feasible via a combination of brief neuropsychological tests and biomarkers in an at - risk cohort . In this subcohort from adapt, measures of global cognitive function (3ms), episodic memory (hvlt - r trial 4), language fluency, and serum a142/a140 ratio achieved an excellent accuracy of 91% . Furthermore, sensitivity with specificity of at least 80% for the combined measures was superior to neuropsychological measures or to serum a levels alone . We have recently shown that a levels alone can predict mci / ad, but a levels are influenced by vascular disease and associated medications and require adjustment to observe the full impact of a in predictive modeling . We have also shown that in subjects diagnosed with ad, there is an association between measures of language tests of fluency and object naming and a140 and that memory performance is associated with serum a142 . An association between serum a140 and cognitive measures of memory and language has also been reported in cognitively normal older adults . High baseline a142 and a140 with stable a142 over time is shown to be associated with diminishing cognition . More recently, yaffe and colleagues demonstrated that low a142/a140 ratios predict cognitive decline over 9 years . In our study, we demonstrate that low a142 and a142/a140 ratios are associated with cognitive decline even within one year . This is extremely valuable from the clinical perspective, as the ability to identify at - risk individuals within a year prior to the onset can significantly improve the quality of care and the recruitment strategy for prevention trials by redirecting those individuals who may not benefit from preventive therapies towards more suitable clinical intervention . This is demonstrated by recent adapt findings, which suggest that individuals with low baseline cognitive scores converted soon after the trial initiated and that neither naproxen nor celecoxib intervention was beneficial to these individuals . Collectively, these findings suggest that combining cognitive tests with blood a may be useful for predicting future mci / ad, which to date has not been explored, particularly as either a or the cognitive tests alone may not have the desired sensitivity or specificity for prediction of future mci / ad . This current work presented here provides evidence that the combination of brief neuropsychological tests and blood a has potential utility in predicting mci / ad at least 2 to 4 years prior to the clinical classification of mci or diagnosis of ad . In addition, our findings also demonstrate the importance of accounting for factors such as apoe, vascular risk factors, and medications when using a in predicting mci / ad . Although at present no studies have reported sensitivity and specificity of csf a142 in predicting mci / ad conversion from normal cognition, a large multicenter study has shown that csf a142 predicts transition from mci to ad, while tau alone achieved a high sensitivity (83%) with acceptable specificity (72%). It is interesting to note that our findings using blood and cognitive tests, a far less invasive method, resulted in higher sensitivities and specificities for predicting cognitive decline in at - risk cognitively normal older adults . Despite the limitation that blood sampling was not conducted at the same time point as the cognitive testing, our data provide strong support for further evaluation of this approach, particularly as we have not seen significant fluctuations in a levels over a one - year period (pers . Our study provides support that blood - based a levels may have diagnostic utility when combined with neuropsychological measures . This proposed method warrants further investigation to determine its practical applicability in specialized clinic setting by allied health personal and in routine primary care clinics.

The wnt/-catenin pathway has been implicated in the development and progression of melanoma and a wide range of cancer types, including colorectal cancer, breast cancer, esophageal carcinoma, and liver cancer [13]. Under normal conditions, increases in expression and binding of certain wnt ligands to frizzled receptor or mutations in specific components of the -catenin degradation assembly deactivate this regulatory mechanism . Nuclear -catenin stimulates transcription of a large number of tcf/-catenin responsive genes that include cyclin d1, c - myc [5, 6], and the melanocyte - specific gene, microphthalmia - associated transcription factor mitf - m . Thus, accumulation of nuclear -catenin as observed in several cancer types is considered a marker of canonical wnt/-catenin pathway deregulation and unfavorable prognosis [3, 8]. Previous studies have reported an association between nuclear -catenin accumulation and melanoma progression and suggested nuclear -catenin to be a marker of poor prognosis [1, 7]. However, recent studies have shown that contrary to breast and colon cancer, metastatic progression of melanoma is associated with decreases in nuclear and cytoplasmic -catenin expression [9, 10]. Moreover, clinical, genetic, and histological studies suggest that nuclear and cytoplasmic -catenin may be used as biomarkers of good prognosis in melanoma [1114]. Recently, hhr6, a human homologue of the yeast rad6 gene and a principal component of the postreplication dna repair pathway, has been identified as an important regulator of canonical wnt/-catenin signaling [15, 16]. Hhr6, referred hereafter as rad6, stabilizes -catenin by polyubiquitin modifications that render -catenin resistant to 26s proteasomal degradation . Furthermore, rad6 is a transcriptional target of -catenin, thus revealing a positive feedback loop between -catenin - mediated activation of rad6 gene expression and rad6-induced -catenin stabilization . Rad6 expression is low in normal breast tissues; however, increases in rad6 protein expression are detected in hyperplastic, ductal carcinoma in situ (dcis) and invasive breast carcinomas . We have previously demonstrated a role for rad6 in breast cancer progression through its regulatory effect on the canonical wnt/-catenin pathway . Since the decrease / loss of nuclear -catenin [9, 10], rather than increases as in breast cancer, is linked to melanoma progression, it is not known whether rad6 and -catenin work in concert to promote melanoma pathogenesis . Furthermore, rad6 expression in the skin has not been investigated, and there are no data on the role of rad6 in the pathogenesis of benign (nevi) and malignant (melanoma) melanocytic lesions . It is important to address this gap in knowledge because of the unmet medical need for new effective antimelanoma therapies and because rad6 and -catenin have been identified as therapeutic targets [19, 20]. In this study, we examined rad6 and -catenin expressions in serial sections of nevi, primary, and metastatic melanomas to determine their potential roles in melanoma development and metastatic progression . Our data suggest that membranous relocation of -catenin and upregulation of rad6 are independent markers of melanoma development and progression . We also offer a hypothesis that explains the role of membranous -catenin relocation and decreasing cytoplasmic -catenin in melanoma development, a phenomenon that has been linked to unfavorable prognosis [9, 21, 22]. Cases were retrieved from the files of the pinkus dermatopathology laboratory (pdl), a private dermatopathology laboratory located in monroe, mi . Preserved paraffin - embedded tissue specimens collected for each case were assigned an accession code that excluded patient identifier information . Nevus and primary melanoma cases were selected for study using random numbers generated by a uniform random number generator (stata / mp 13.1). The study groups consisted of 30 cases of melanocytic nevi, 29 cases of primary cutaneous melanoma, and 29 cases of metastatic cutaneous melanoma . The study includes all metastatic cutaneous melanoma samples that were archived between 2010 and 2012 . The number of cases for each nevus and primary melanoma subtype was determined to reflect the lesion's relative representation in cases obtained at the pdl during the above period . Atypical nevi were diagnosed using criteria originally proposed by clark and lesion architecture as reviewed by roth et al . . Primary antibodies used in the study are as follows: (i) anti--catenin (is702) was purchased from dako (glostrup, denmark) and used in an undiluted form; and (ii) anti - rad6 (ab31917) was purchased from abcam (cambridge, ma) and used at a 1: 500 dilution . In humans, the yeast homologous rad6 gene is duplicated and the proteins encoded by the two genes hhr6a (or rad6a) and hhr6b (rad6b) from chromosomes xq24-q25 and 5q23-q31, respectively, share 95% identical amino acid residues . Neither ab31917, our own rad6 antibody, nor any other commercially available anti - rad6 antibody is currently able to distinguish between rad6a and rad6b proteins . Therefore, rather than referring as rad6a or rad6b, we refer to the protein detected by the antibody as rad6 . Briefly, five - micrometer sections were deparaffinized in xylene and rehydrated in graded ethanol . For antigen retrieval, sections were microwaved in citrate buffer ph 6.0 (biogenex, san ramon, ca, usa) for 12 min at 95c and cooled for 30 min prior to immunostaining . Sections were incubated with 3% hydrogen peroxide for 15 min, followed by incubation with primary antibody for 60 min . An automated immunostainer (i6000; biogenex) was utilized for subsequent incubation steps: sections were incubated in multilink biotinylated anti - igg for 20 min, horseradish peroxidase conjugated secondary antibody for 20 min, followed by development with 3-amino-9-ethyl - carbazole for 10 min (biogenex). All incubation steps were performed at room temperature, and sections were washed with tris - buffered saline between incubations . Lung and colon cancer tissues were included as positive controls for immunostaining with anti--catenin antibody, and breast cancer tissues were included as positive controls for staining with anti - rad6 antibody . Stained sections were independently enumerated by two coauthors (d. r. mehregan and m. campbell), who were blinded to patient medical records for each case . Blinded enumeration was performed under light microscopy at 400x magnification, and an ocular grid consisting of a simple square lattice of 100 test points was utilized to count the number of positively and negatively stained melanomas or nevus cells for each section . Per section, the total number of positively and negatively stained cells was counted for each of three sequential horizontal fields . The mean value of the three fields was used to estimate the relative density of cells in a specimen . To increase assessment accuracy, all positively and negatively stained melanomas or nevus cells in a visual field when independent readings for positively stained cells differed by 20% for a given section, both evaluators reviewed the section together to establish a consensus reading . A specimen was considered negative if less than 4% of the cells were immunostained for rad6 or -catenin . A tumor was considered to be stained with high intensity if> 50% of the cells in a specimen expressed rad6 or -catenin, similar to the criteria used by mineta et al . . Kruskal - wallis tests were used to compare groups on the basis of continuous variables such as age and percent positive cells . Chi - square tests for differences in proportions were used to compare groups on the basis of categorical variables such as gender and -catenin localization . Spearman's rank correlation was used to assess the pairwise association between age and percent of rad6 positive and -catenin positive cells . Multinomial logistic regression was used to assess the simultaneous association of rad6 and age with diagnostic group . Adjustments were made for multiple comparisons using wilcoxon rank sum tests with bonferroni correction for pairwise comparisons . Our analysis included 30 individuals diagnosed with nevi, 29 with primary melanoma, and 29 with metastatic melanoma (table 1). These groups differed marginally with respect to gender (p = 0.08) and significantly in age (p = 0.0001). Significant age differences were observed between individuals with nevi and those with either primary melanomas (p = 0.02) or metastatic melanomas (p = 0.0001). In contrast, no age difference is observed between individuals with primary and metastatic melanomas (p = 0.27). Significant differences in age were also observed between groups defined by -catenin localization (p = 0.007). Individuals with -catenin localized in the cytoplasm were significantly younger than individuals with -catenin localization at the cell membrane (p = 0.02) and marginally younger than individuals with -catenin localization at both the cytoplasm and the cell membrane (p = 0.05). When age was categorized as <50, 5060, and> 60 years, there were statistically significant differences in rad6 expression between the groups (p = 0.0008), although there is substantial variability (table 2). Median rad6 is greater in the group of people older than 60 years compared to the 5060 years old group (p = 0.04) and <50 years old group (p = 0.001). A 20% discrepancy of positively stained cells between the two evaluators was observed in fewer than 5% of the cases, and these cases were evaluated together to establish a consensus reading . Melanoma development and progression were not associated with significant changes in percentage of specimens expressing -catenin . -catenin staining was observed in 97% of nevi and in all primary and metastatic melanomas . Also, the percentages of nevi (93%), primary melanoma (97%), and metastatic melanoma (93%) that expressed -catenin in more than 50% of the cells did not differ significantly (figures 1, 2, and 3). However, significant differences were observed between the percentages of nevi (59%), primary melanoma (90%), and metastatic melanoma (56%) that expressed -catenin in more than 90% of the cells (p = 0.02; figure 2). These differences were greatly impacted by the percentage of primary melanomas (48%) that expressed -catenin in 100% of the cells, which was approximately twofold higher than the percentages of nevi (21%) or metastatic melanoma (26%) (data not shown). None of the nevi or melanomas expressed -catenin in the nucleus (figure 1). The percentage of tumors that expressed membranous -catenin increased dramatically from nevi (10%) to primary and metastatic melanomas (83% and 93%, resp . ; concurrently with this increase, the percentage of tumors that expressed cytoplasmic -catenin decreased from nevi (90%) to primary and metastatic melanomas (45% and 38%, resp . ; in contrast, no significant differences were observed between the percentages of primary and metastatic melanomas that expressed -catenin at either the membrane (83% and 93%, resp . ; p = 0.289) or the cytoplasm (45% and 38%, resp . ; p = 0.633; figure 4). While all four nevi types (junctional, intradermal, compound, and atypical) expressed -catenin in the cytoplasm, only junctional and atypical nevi expressed -catenin at the plasma membrane . As opposed to nevi types, the three primary melanoma types examined (superficial spreading, nodular, and lentigo maligna) did not differ in -catenin localization as -catenin was localized at the plasma membrane, in the cytoplasm, or in both (figure 4). Conversely, all primary melanomas (100%) and the majority of metastatic melanomas (96%) exhibited greater than 50% rad6 expression . The increase in tumor populations expressing rad6 from 37% of nevi to 100% of primary and metastatic melanomas was significant (p = 0.0001; figures 1, 2, and 3). Melanoma progression from primary to metastatic disease was not associated with changes in the (i) percentage of melanomas expressing rad6 (100% of primary and metastatic melanomas), or (ii) percentage of melanomas expressing rad6 in more than 50% of the tumor cells (100% and 96% of primary and metastatic melanomas, resp . ). The increase in proportion of tumor populations, expressing rad6 in more than 50% of the cells in primary melanoma (67%) versus metastatic melanoma (79%) was not significant (p = 0.37; figure 2). This study was not designed to test whether the distribution of the tumor cells positive for rad6 is the same between the subtypes of nevi . However, the percentages of benign tumors that lacked rad6 were similar between atypical nevi (62%) and the group of other three nevi types (59%). Interestingly, only one of the 30 nevi (atypical nevus, 3%) expressed rad6 in> 80% of the cells, and none of the primary and metastatic melanomas expressed rad6 in <40% of the cells (figure 2). These results prompted us to examine whether rad6 expression can serve as a marker for histological diagnosis of melanoma . Using a multiple logistic regression model, we found that the strength of rad6 expression is a strong predictor of melanoma (p <0.001) even when age group (p = 0.65) and gender (p = 0.24) are included in the model . The model predicts that every 1% increase in rad6 expression results in a 9% increase in the probability that a lesion is melanoma . If we assume that a predicted probability of> 0.5 indicates melanoma, the model with only rad6 has sensitivity of 93% and specificity of 80% . These results are very encouraging; however, they need to be validated in a larger study . The expression profiles of -catenin and rad6 differed considerably in nevi . Approximately 93% of nevi expressed -catenin in more than 50% of the cells, whereas only 27% of the same population of nevi expressed rad6 (figure 2). -catenin and rad6 expressions in nevi were not significantly correlated (r = 0.06; p = 0.77). There is a 2.7-fold difference in the percentage of primary melanomas (100%) expressing rad6 compared to nevi (37%) and virtually no difference in -catenin expression between primary and metastatic melanomas (100%). Accordingly, rad6 and -catenin expressions in primary melanoma were not correlated (r <0.001, p> 0.99). A significant correlation between rad6 and -catenin positive cells however, this association diminished (r = 0.40, p = 0.05) following the exclusion of two observations which are disproportionally influential (one with <50% positive rad6 and one with <50% positive -catenin). This is the first study to characterize rad6 expression in cutaneous benign and malignant melanocytic tumors . In this study, we examined the association between rad6 and -catenin expressions in benign and malignant melanocytic tumors to determine whether rad6 works in concert with -catenin to influence melanoma development and progression . Rad6 and -catenin positively regulate each other in breast cancer [15, 18]. However, while -catenin has been implicated in the pathogenesis of melanoma and other cancer types, data about the role of rad6 in cancer pathogenesis are mostly limited to breast cancer . Therefore, we hypothesized that comparison of rad6 and -catenin expressions in the same nevi and melanoma tumors would help determine whether these two signals collaborate to promote melanoma development and progression as they do in breast cancer [15, 28]. Accumulation of nuclear and cytoplasmic -catenin has been implicated in driving the development and progression of several cancer types (e.g., colon and ovarian cancers) [2931]. However, our results show that the expression levels of -catenin do not contribute to melanoma initiation and progression since no difference in -catenin levels was found between nevi, primary melanoma, and metastatic melanoma (93%97% of all samples expressed -catenin in> 50% of the tumor cells). The high expression levels of -catenin are in line with the crucial role of -catenin in differentiation and proliferation of both normal melanocytes and metastatic melanoma cells . Also, our findings are in agreement with previous reports of positive -catenin staining in nevi (100%) and primary melanoma (95%, 94%) but are higher than reported in metastatic melanoma (75%, 68%) [9, 21]. The variation in expression of -catenin levels in metastatic melanomas between the studies while we studied only melanoma metastases to the skin, other studies either obtained 58% of their specimens from lymph nodes, tonsil, and liver or did not identify the anatomical site of their metastases [9, 21]. Furthermore, different anatomical sites may regulate dissimilar antigen expressions in metastases that originate from the same primary tumor in the same patient [33, 34]. Previous studies have shown higher percentages of nuclear -catenin in nevi than in melanoma (84% versus 33%, and 44% versus 15%) [9, 22]. Those observations provided the basis for the currently held concept that loss of nuclear and cytoplasmic -catenin suggest poor prognosis and decreased overall survival of melanoma patients [12, 22]. In light of these data, the absence of nuclear -catenin in all the nevi and melanomas analyzed in our study was surprising . Usage of different anti--catenin antibodies may explain in part the discrepancy in nuclear -catenin expression observed between the studies . However, our results are consistent with the lack of nuclear -catenin reported in four studies which comprised 57 nevi, more than 55 primary melanomas, and 20 metastatic melanomas [21, 3537]. Moreover, nuclear -catenin was not found in either the nevus portion or the melanoma portion of 15 cutaneous lesions and was absent in additional 42 primary melanomas . In another study of 70 primary melanomas, nuclear -catenin was reported in only 6.4% of the melanomas . Finally, in a study of 230 primary and metastatic melanomas, nuclear -catenin was reported in only 13 cases (5.6%) and therefore those cases were excluded from analysis . Taken together, the absence or negligible amount of nuclear -catenin detected in the aforementioned studies as well as ours suggests possible extranuclear roles for -catenin in nevi and melanoma . This notion is supported by a role for cytoplasmic -catenin to execute functions that do not require nuclear translocation (e.g., activation of map kinase p38 and nf - kb) [37, 40]. A major finding of this study is the association between melanoma development and intracellular redistribution of -catenin . The percentage of cases that expressed -catenin on the cell membrane increased dramatically from 10% in nevi to 83% and 93% in primary and metastatic melanomas, respectively . Concurrently, the percentage of cases that expressed cytoplasmic -catenin decreased from 90% in nevi to 45% and 39% in primary and metastatic melanomas, respectively (figure 4). We hypothesize that the relocation of -catenin from the cytoplasm to the cell membrane may serve as a deactivating mechanism of canonical wnt/-catenin signaling and that the resulting reduction in cytoplasmic -catenin level may contribute to the malignant transformation of melanocytic nevi . The proposed hypothesis is supported by the following observations: (i) as in our study, bachmann et al . Also reported an association between nevus to melanoma development and relocation of -catenin to the cell membrane . Nevertheless, the authors of that study did not offer an explanation for their observation; (ii) our analysis of the data of kagashita et al . Showed -catenin decrease in the cytoplasm and increase at the cell membrane and that these changes in -catenin distribution corresponded with the malignant transition of nevi; (iii) wnt4 signal has been identified as a mechanism that can drive -catenin relocation from cytoplasm to cell membrane; and (iv) -catenin relocation from cytoplasm to cell membrane has been reported to block -catenin signaling in a human embryonic kidney (hek293) cell line . Of note, this hypothesis can explain how despite the abundant -catenin expression in melanoma [1, 7], cytoplasmic -catenin is selectively decreased, a phenomenon that has been associated with unfavorable melanoma prognosis [9, 21, 22]. Our current efforts are directed towards determining if the increases in membranous -catenin observed in primary and metastatic melanomas result from relocation of existing molecules in the cytoplasm or deposition of newly generated -catenin at the membranous site . Rad6 has been implicated in early breast cancer development since an increase in rad6 levels is observed in adenosis and benign hyperplasias as compared to normal tissue . In contrast, our findings do not support rad6 to play a similar role in nevus formation as in benign breast neoplasia, since 63% of the nevi were negative for rad6 . Rad6 has also been implicated in breast cancer progression because rad6 levels increase with progression from ductal carcinoma in situ to invasive primary carcinoma and metastatic cancer [15, 28]. In accordance with the upregulation of rad6 in early stages of breast cancer development as compared to benign hyperplasia [15, 17], we observed a striking increase in rad6 expression in primary melanoma when compared to nevi . While all primary melanomas displayed strong rad6 staining (> 50% of the tumor cells), rad6 was negative in 63% of the nevi . These findings suggest that rad6 may play a role in malignant transformation of nevi as in breast cancer . Progression of melanoma from primary to metastatic disease was not significantly associated with further changes in the percentage of tumors expressing rad6 or rad expression intensity as> 50% of tumor cells stained positively in 100% and 96% of primary melanomas and metastatic melanomas, respectively . These findings suggest that rad6 may play a sustained role in melanoma metastasis as it does in melanoma development . In benign and malignant breast tumors, rad6 stabilizes -catenin, and, in turn, -catenin positively upregulates rad6 transcription [1517]. However, this direct positive correlation between -catenin and rad6 expression does not appear to be conserved in melanoma as the expression profiles of -catenin and rad6 differed considerably in nevi . Approximately 93% of nevi expressed -catenin compared to only 27% of nevi that expressed rad6 in more than 50% of their cells (figure 2). These observations suggest that the high -catenin expression in nevi is likely driven by regulators other than rad6 . At first glance, it would appear that -catenin and rad6 expressions are correlated in primary melanoma because these proteins were coexpressed in approximately all primary melanomas . Also, these findings correspond to the 80% correlation between rad6 and -catenin expressions in primary breast cancer . However, it is unlikely that the high rad6 expression in primary melanoma is driven by the concurrent high -catenin expression, because rad6 expression is low in nevi despite the presence of high cytoplasmic -catenin expression that is comparable to primary melanoma . This notion is confirmed by lack of statistical correlation between rad6 and -catenin expressions in primary melanoma . -catenin is not the only activator of rad6; for instance, rad6 is activated by nerve growth factor in nervous tissue . Therefore, it is conceivable that, in primary melanoma, rad6 expression is regulated by yet unidentified activators . We also demonstrated that further progression of melanoma from primary to metastatic disease is not associated with a correlation between the -catenin and rad6 expressions . Taken together, our study does not support a direct positive interaction between -catenin and rad6 in either benign or malignant melanocytic tumors . We characterized for the first time rad6 expression in cutaneous benign and malignant melanocytic tumors . We are showing a striking upregulation of rad6 from a negative expression in most benign melanocytic tumors to 100% of primary and metastatic melanomas . These findings strongly suggest a role for rad6 in the development of primary melanoma and metastatic disease . We show that in contrast to rad6, -catenin is expressed in more than 50% of the tumor cells in almost all nevi and melanoma tumors . Taken together, in contrast to the rad6 -catenin positive relationship in breast cancer [1517], our study does not support a similar positive interaction between -catenin and rad6 in benign or malignant melanocytic tumors . Finally, our findings suggest a role for the cytoplasmic to membrane translocation of -catenin in the development of primary melanoma . Future studies will determine whether newly generated -catenin at the membranous site coincide with -catenin translocation from the cytoplasm.

The guanine heterocyclic ring is rich in chemical reactivity toward oxidants and adduct - forming species . In dna, 2-deoxyguanosine (dg, mass = m) is the chief site for base oxidation due to its low redox potential leading to many products that are characterized by their mass signatures . The principal products characterized with one - electron oxidants in aqueous solutions can be grouped based on the site of reaction on the radical intermediate resulting from one - electron oxidation of dg . The 2-deoxyribonucleoside products arising from initial reactivity at c5 of dg include a four - electron oxidation product, an imidazolone (diz, m-39) that hydrolyzes to an oxazolone (dz, m-21), and the two - electron oxidation product 5-carboxamido-5-formamido-2-iminohydantoin (d2ih, m+34; scheme 1). When the initial reaction occurs at c8 of dg, either 2,6-diamino-4-hydroxy-5-formamidopyrimidine (fapy - dg, m + 18) is observed under reducing conditions or 8-oxo-7,8-dihydroguanine (dog, m+16) is observed under oxidizing conditions, in which the latter compound is a key marker for monitoring oxidative stress in cells (scheme 1). The two - electron oxidation product dog is stable but is highly susceptible to further oxidation leading to two hydantoin compounds . The yield for the thermodynamically preferred product spiroiminodihydantoin (dsp, m + 32) is greatest in nucleoside reactions at ph> 6 or in unencumbered reaction contexts (i.e., single - stranded dna or g - quadruplexes), while the yield of 5-guanidinohydantoin (dgh, m + 6) is greatest in nucleoside reactions at ph <6 or in sterically demanding duplex contexts (scheme 1). The hydantoins are also readily formed from direct four - electron oxidation with o2 . This list of products represents those that are consistently observed from many oxidant systems conducted by several laboratories; however, other compounds have been reported in lower yield . In the cellular context, oxidation reactions with dg can have other nucleophilic participants such as amines or phenols . In most cases, when amines or phenols participate in the reaction, products are observed with similar backbone structures as characterized with water serving as the nucleophile . For example, when dg is oxidized in the presence of lysine, products retaining the dg heterocycle and those with a core similar to dgh or dsp have been observed . The adducts characterized by our laboratory and others show lysine competing with water for sites of covalent bond formation, where a single lysine is observed at c8 analogous to dog, at either c5 or c8 of a spirocyclic core similar to dsp, and last a bis - adduct at c5 and c8 with a spirocyclic ring structure has been identified (figure 1). The lysine adducts are all stable allowing their quantification and characterization . As a last example, the polyamine spermine has been adducted to dg and dog under oxidative conditions . In the dg oxidation studies, a spermine adduct at c8 is observed retaining the dg heterocyclic core, while dog oxidations yield an adduct at c5 that does not undergo acyl migration to create a spirocycle, but instead the polyamine can generate an unstable hemiaminal intermediate that decomposes leaving a ribosylurea lesion at the adduct formation site . Moreover, the facile formation of spermine adducts to dog oxidation intermediates has been harnessed for quantification of dog from dna samples . In contrast to amines, phenols (i.e., tyrosine) are more redox active than amines and better able to compete with dg for oxidant during reactions . The products observed when phenols participate with dg during oxidation have more variability in their structures compared to amine adducts . These model studies aid in understanding the chemical nature of dna protein cross - links that are detrimental to cellular processes . In the current work, oxidations that allowed dg to react with nh3 were conducted while the full product and diastereomer distributions were monitored . After careful characterization of the nucleoside reactions, similar oxidations were conducted in the single - stranded and duplex oligodeoxynucleotide (odn) contexts . The purified ammonia adducts were also studied with respect to their decomposition pathways leading to end products that have not been previously determined . Lastly, polymerase insertion studies were conducted to test the hypothesis that amine adducts might have altered base - pairing preferences; every substitution of an oxo group on a base for an amino group converts a hydrogen bond acceptor into a potential hydrogen bond donor . These results provide a fundamental understanding of the stability and base - pairing properties of amine adducts to dg that result during oxidation, which may occur in vivo due to the high concentration of nucleophilic amines in the vicinity of the genome . Adducts observed when dg and lysine are allowed to react in the presence of oxidant . The unencumbered nucleoside dg (1 mm) was chosen for the initial oxidation reactions where nh4cl (20 mm) provided the source of nucleophilic nh3 . All reactions were conducted with 75 mm napi buffer at ph 7.4 at 22 c . The oxidants chosen include the photooxidants riboflavin and rose bengal and the one - electron oxidant na2ircl6 . Reactions were conducted in triplicate to achieve 70% conversion to products in each reaction, and the products (scheme 2) were analyzed by a dual hplc method . The first round of hplc analysis utilized a reversed - phase column to identify dog (m+16) and 8-amino - dg (m+15), while all other products eluted in the void volume . The void volume from the previous run was collected and analyzed on a hypercarb hplc column that allowed analysis of the hydantoins, ammonia adducts, and dz . However, diz hydrolyzes to dz that was detected on the hypercarb column; thus, formation of diz is inferred from quantification of dz . Moreover, the hypercarb hplc column also allowed separation and quantification of all spirocyclic diastereomeric pairs of products . The dgh diastereomers are interconvertible, and thus, their diastereomer ratios are not reported (see the supporting information for complete experimental details). Lastly, a test reaction with na2ircl6 was conducted in which half was directly analyzed by the hypercarb hplc column and the other half was analyzed by the dual hplc method outlined above . The product distributions observed from these comparative studies were within 3% of one another . The photooxidant riboflavin led to the largest number of different products and was used to obtain suitable amounts of material for characterization . Esi - ms (figure s1, supporting information), and the adducts observed that did not involve participation of nh3 during product formation included dz (m - 21), dog (m + 16), dgh (m + 6), and dsp (m + 32, scheme 2). Confirmation of their structures was achieved by esi - ms / ms fragmentation of the free bases while monitoring the daughter fragments . The esi - ms / ms experiments were conducted on hplc purified nucleosides in which the n - glycosyl bond was cleaved in the ionization source to liberate free bases that were further fragmented in the cid chamber to generate the daughter fragments used in identification of the structures . The ms / ms fragmentation spectra were compared to literature values for the two dsp diastereomers (figure 2) and dz to further confirm their structural assignments (figures s2 and s3, supporting information). The structure for dsp has been further established by x - ray crystallography and nmr . Ammonia adducts observed included two pairs of chromatographic peaks for the diastereomers of spirodi(iminohydantoin)-2-deoxyribonucleosides (dsi), whose names are based on the site of nh3 attachment to guanine, thus 5-dsi (m + 31) and 8-dsi (m + 31) (scheme 2, see reference cited for the correct dsi iupac nomenclature). To confirm the identities of the 5- and 8-dsi constitutional isomers and their respective diastereomers, esi - ms / ms of the free bases were conducted (figure 2). Comparison of the esi - ms / ms spectrum for 5-dsi with dsp gave a pair of peaks for 5-dsi that established c5 as the site of nh3 attachment (m / z [m + h] = 140 and 96, figure 2). As for 8-dsi, comparison of its esi - ms / ms spectrum with those obtained from dsp and 5-dsi identified new masses that are best explained by nh3 attachment at c8 (m / z [m + h] = 141 and 97, figure 2). On the basis of the proposed mechanism (scheme 2), a product that includes bis - addition of nh3 at c5 and c8 of an oxidized guanine to yield a spirocycle (5,8-dsi, m+30) is possible; however, this adduct was not observed for reasons that will be elaborated on below . Additionally, nh3 adducts with a dgh core were not observed most likely due to the fact that the reactions were performed at ph 7.4 where yields of dgh are minimal . Lastly, the yields of dog (m + 16) and 8-amino - dg (m + 15) were very low (<1%) as determined by lc - esi - ms (figure s1, supporting information); hence, their quantities are not reported . The nucleosides dog and c8 amine - adducted dg have redox potentials that are 600 mv below the parent nucleoside dg causing them to be much more susceptible to further oxidation . Further, one - electron oxidized dog was observed to have an even lower redox potential than the parent compound dog, which means that once oxidation of dog, and likely 8-amino - dg, occurs product formation is inevitable due to the 70% conversion to product that yielded the spirocyclic compounds dsp, 5-dsi, or 8-dsi . Esi - ms / ms spectra for dsp (top), 5-dsi (middle), and 8-dsi (bottom). The data provided was collected on the first eluting diastereomer of each spirocycle from a hypercarb hplc column . Data for the second eluting diastereomer for dsp, 5-dsi, and 8-dsi can be found in the supporting information (figures s2, s4, and s5). The heavy lines represent the fragment observed and the thin lines represent the portion of the molecule lost upon fragmentation . Relative product distributions observed with each oxidant system were determined by integration of the hplc peak areas measured at 240 nm followed by normalization via each molecule s unique extinction coefficient (240 nm). Extinction coefficients for 5-dsi and 8-dsi are not known but were determined from experiments that are reported below . In the riboflavin - mediated oxidations, this result was anticipated because riboflavin is a type i photooxidant and effects oxidation by electron transfer from dg yielding o2 under aerobic reaction conditions . Oxidation of dg by one - electron and proton transfer yields an intermediate radical (dg) that only couples with o2 to initially yield diz that is prone to hydration leading to dz (scheme 2), the species quantified . The adducts derived from the nucleophile trapping of electrophilic dg oxidation intermediates show nh3 participation through the products 5-dsi (29%) that was the highest yielding nh3 adduct followed by nearly 3-fold less 8-dsi (11%). The participation of h2o as the trapping nucleophile was identified by the lower yields of dsp (11%) and dgh (4%). The nucleophilicity of nh3 is much greater than h2o; therefore, as expected, adducts resulting from nh3 participation dominated over those derived from h2o . Relative product distributions observed when dg reacted with nucleophilic h2o or nh3 in oxidation reactions . The oxidants include the photooxidants riboflavin and rose bengal, as well as na2ircl6 . Reactions were conducted with 1 mm dg, 20 mm nh4cl, in 75 mm napi buffer (ph 7.4) at 22 c . (1) photoactivation of riboflavin (200 m) was achieved with 350 nm light for 3 h, (2) photoactivation of rose bengal (100 m) was achieved with 350 nm light for 3 h, and (3) oxidation with na2ircl6 (10 mm) was achieved by bolus addition of the salt . Results represent the average of triplicate trials and the error is 10% of each reported value . The second oxidant studied was rose bengal, a type ii photooxidant (o2) that furnished 5-dsi as the major product (62%) followed by a 3-fold lower amount of dsp (31%), and low yields of dz (5%) and dgh (2%) were observed to complete the mass balance (figure 3). As expected, this oxidant did not yield 8-dsi based on its mechanism of oxidation . Oxidation of dg by o2 proceeds by 4 + 2 cycloaddition to the imidazole ring, followed by ring opening to yield 8-hoo - g that eliminates water giving the proposed electrophile dog . Next, dog is trapped by nucleophiles at c5 leading to dsp with h2o or 5-dsi with nh3 (scheme 3). Support for exclusive nucleophilic attack at c5 under o2 oxidations was derived from h2o studies followed by mapping the labeled site by esi - ms / ms; the current observation that the only nh3 adducts were the 5-dsi diastereomers further support this previously proposed mechanism, and further confirms our structural assignments for these peaks . Comparisons of the product distributions from the photooxidants riboflavin and rose bengal show a dramatic difference with respect to the major product observed (figure 3). In the riboflavin oxidation, dz was the major product while in the rose bengal oxidation, 5-dsi was the major product, and these observations can be ascribed to each oxidant s unique mechanism of oxidation (schemes 2 and 3). Product distributions from this oxidant included nearly equivalent distributions of dsp (50%) and 5-dsi (42%) with the mass balance completed by dgh (8%, figure 3). In this oxidation reaction, the distribution did not yield nh3 adducts as the major products, which was unexpected on the basis of nh3 being the better nucleophile compared to h2o . Comparison of the product distributions observed with the one - electron oxidants riboflavin and na2ircl6 identified both nh3- and h2o - adducted compounds . The current results differ from those previously reported by our laboratory when lysine was adducted to dg in analogous oxidation reactions . Previously, in riboflavin oxidations spirodihydantoins with lysine at c8 were 2-fold greater than those with lysine at c5 (figure 1), and in na2ircl6 oxidations a c8 lysine adduct was observed . In contrast to these results, riboflavin oxidations in the presence of nh4cl gave more c5 adducts (29%) than c8 adducts (11%), and na2ircl6 oxidations did not yield detectable amounts of c8 adducts . We propose the difference in the current ammonia results compared to the lysine data can be attributed to a difference in the mechanism of product formation for the c8 adduct in the nucleoside context . The former work from our laboratory proposed that c8 amine adducts result from oxidation of the amine to the aminyl radical that adds at the c8 carbon of dg followed by oxidation leading to product formation (scheme 4). In nucleoside dg, this proposed mechanism best supports the observation of ammonia adducts at c8, because upon one - electron oxidation of dg the initial radical cation (dg) formed is very acidic (pka 3.9) and rapidly deprotonates to the neutral radical (dg) that is not susceptible to nucleophilic attack . Thus, dg reacts with o2 yielding diz / dz and not with amines or h2o . Formation of amine adducts at c8 must result from a difference in the amine reactivity . The key difference between lysine and ammonia resides in their standard reduction potentials . In general, primary amines (1.0 v vs nhe, ph 10) have a lower redox potential than ammonia (> 1.3 v vs nhe, ph 9); this trend should scale down to ph 7, in which the oxidations were conducted . Thus, oxidation of lysine to an aminyl radical that adds to c8 of dg is possible with na2ircl6 (0.9 v vs nhe, ph 7) and riboflavin (1.7 v vs nhe, ph 7); in contrast, the analogous reaction does not readily occur for nh3 because at ph 7 dg is the dominant site of oxidation due to its lower redox potential leading to products other than 8-dsi . In summary, one - electron oxidant driven oxidations of dg in the presence of nh3 lead to spirocyclic adducts at c5 and c8 that are in competition with h2o adducts of the same core structure . As the next step, we set out to determine the decomposition products and pathways through which the ammonia adducts proceed . Hplc was used to provide diastereomerically pure 5-dsi and 8-dsi samples that were subjected to conditions of ph 3 (0.1% formic acid) or ph 10 (20 mm napi) at 22 c for 30 min or 10 h. not only could the decomposition products be determined, but having diastereomerically pure starting material also allowed us to probe the mechanism of decomposition, specifically for 5-dsi . First, both 5- and 8-dsi were stable at ph 10 during this time frame, while 5-dsi readily hydrolyzed to dsp at ph 3 in 30 min and 8-dsi hydrolyzed to dsp after 10 h. for 5-dsi, deamination of the amine group can occur by two possible mechanisms: (1) a retro - acyl migration can occur followed by loss of nh3 to yield an electrophilic intermediate (dog) susceptible to h2o attack followed by a second acyl migration back to dsp or (2) acid - catalyzed deamination can occur directly to furnish dsp (scheme 5). If the retro - acyl migration proceeds on the diastereomerically pure sample, it is expected that a mixture of dsp diastereomers would be observed, and if direct deamination occurs, then only one dsp diastereomer would be predicted . Upon incubation of one diastereomer of 5-dsi at ph 3, only one diastereomer of dsp was observed, supporting the direct deamination mechanism . Moreover, the early eluting 5- and 8-dsi nh3 adducts decomposed to the early eluting dsp isomer and the late eluting adducts deaminated to the later eluting dsp isomer (figure s6, supporting information). These results aid in establishing the absolute configuration of the 5-dsi and 8-dsi diastereomers that will be discussed below . Moreover, these results also allude to an explanation as to why the bis - ammonium adduct was not observed in any of the reactions . Based on these data, the bis - ammonium adduct, if formed, could rapidly deaminate to yield either 5-dsi or 8-dsi, although this hypothesis could not be further validated . Utilization of the hypercarb hplc column for analyzing the product distributions of these spirocycles allowed determination of the diastereomer ratios . It has previously been determined that (r)-dsp elutes first from this column and (s)-dsp elutes second . The absolute stereochemistry for the diastereomers of the ammonia adducts had not yet been determined . The results of these studies identified (r)-dsp and (s)-dsp yields to be nearly equal . Also, for the 5-dsi and 8-dsi ammonia adducts nearly equal yields for the diastereomers were observed . On the basis of these results, the defining point of the reaction to determine product stereochemistry must not be very sterically impeded in order to lead to such a small diastereomer preference . As stated above, the diastereomerically pure ammonia adduct samples were determined to decompose to give a single diastereomer of dsp; therefore, on the basis of hplc elution order, the absolute configuration for the diastereomers of the two dsi constitutional isomers can be determined . The first - eluting 5-dsi and 8-dsi diastereomers decomposed to the first dsp diastereomer (r), and the late - eluting diastereomer decomposed to the late - eluting dsp diastereomer (s); therefore, for 5-dsi the r diastereomer elutes first and the s diastereomer elutes second, because the r and s assignments are the same between dsp and 5-dsi . In contrast, the r and s assignments for the 8-dsi diastereomers are opposite those of dsp; thus, (s)-8-dsi elutes first and (r)-8-dsi elutes second and from the hypercarb hplc column (figure 4). These examples provide a fascinating case outlining how the movement of ring substituents affects the r and s stereochemical assignments . Assignment of absolute configurations for the diastereomers of dsp, 5-dsi, and 8-dsi based on their elution profile from a hypercarb hplc column and their ecd spectra . The r and s assignments for the diastereomers of 8-dsi are the opposite of those for the dsp and 5-dsi isomers with the same geometric configuration of the spirocyclic ring, due to a change in the cahn ingold the diastereotopic ammonia adducts were then probed by electronic circular dichroism spectroscopy (ecd). Previously, ecd was used in tandem with vibrational circular dichrosim spectroscopy, nmr, and x - ray crystallography to establish the absolute configuration for dsp . The ecd spectra for (r)- and (s)-dsp isomers gave three lobes that were mirror images of one another, as expected (figure 4). Critical for assigning the absolute configuration for dsp was the low energy lobe, in which the s isomer gave a positive rotation at 258 nm, and the r isomer gave a negative rotation at 259 nm . In comparison to these results, the 5-dsi diastereomers gave ecd spectra that were different than dsp but showed a similarity in the low energy lobe (figure 4). For (r)-5-dsp, the lobe at 258 nm gave a negative rotation and (s)-5-dsi gave a positive rotation at 258 nm . The higher energy lobes observed in the ecd for the 5-dsi diastereomers were different than those measured for dsp (figure 4). As a last comparison, the 8-dsi diastereomer ecd spectra were compared to those recorded for dsp (note: 8-dsi and dsp isomers with the same geometric configuration of the spirocyclic ring give opposite r and s assignments due to a difference in the cahn the low energy lobe gave a negative rotation at 262 nm similar to (r)-dsp, and (r)-8-dsi gave a positive rotation similar to (s)-dsp . Furthermore, the 8-dsi diastereomers also gave similar rotations and energies as their analogous dsp diastereomers at the 238 nm lobe, and 8-dsi and dsp gave similar rotations at the highest energy lobe, but the absolute energy was different (i.e., 202 nm for 8-dsi and 211 nm for dsp; figure 4). The similarity in the critical low energy lobe further supports the absolute configuration assignments that were made from the deamination studies monitored by hplc . Future computational studies to model these ecd spectra will help solidify these conclusions, and may address some of the challenges that occurred during modeling of the ecd spectra for the dsp diastereomers . The ability to deaminate the dsi adducts to dsp allowed the determination of the extinction coefficients for 5- and 8-dsi relative to dsp, in which these were the values used to determine the relative yields . Peak areas for identically pure 5-dsi samples were measured before and after incubation in formic acid and the change in peak area measured was used to determine the relative 240 nm compared to the value established for dsp . A similar experiment was conducted for the 8-dsi diastereomers . From these experiments, the 240 nm for the 5-dsi and 8-dsi diastereomers were determined to be 3800 and 3500 lmolcm, respectively, which are both slightly greater than that determined for the dsp diastereomers (3300 lmolcm). The next step from the nucleoside studies was to explore the context dependence of nh3 adduct formation in single- and double - stranded odns (ssodn and dsodn). The photooxidant riboflavin was chosen for the studies because it gave the most diverse distribution of products in the nucleoside reactions and might provide the most insight into context effects . For these studies, the ssodns selected for analysis were the 18-mers odn-1 and odn-2, and the dsodn context was studied via the duplex formed from these two single strands (odn-12). After oxidation of these odn systems, they were digested with a suite of nucleases and phosphatase to the nucleosides followed by analysis using the previously described hplc methods . Previously, our laboratory demonstrated that the digestion method used provides complete degradation of dsp - containing odns to nucleosides; it is assumed that the dsi adducts are equally digested to completion . The nuclease digestion conditions were modified to include ammonium salts for buffer to prevent loss of 5-dsi via deamination to dsp (scheme 5), controls conducted with sodium salts for buffers did not detect 5-dsi due to deamination to dsp during the nuclease digestion (18 h at ph 5.4, scheme 5). A comparison between the contexts studied when dg was allowed to react with nh3 in the presence of the photooxidant riboflavin is provided in figure 5 . The first observation from these data was with respect to dz distributions that dramatically decreased when proceeding from the nucleoside to odn contexts; specifically, the dz distribution in the dsodn context (2%) was 10-fold less than the ssodn context (20%) and 20-fold less than that observed in the nucleoside context (40%). This observation has already been reported in the literature and is proposed to result from the odn context increasing the lifetime of the dg that reacts with nucleophilic h2o to give the c8 product dog while decreasing the lifetime of dg that reacts with o2 to yield dz (schemes 2 and 6). With respect to the yields of the hydantoins, more dsp relative to dgh was observed in the nucleoside context (11% vs 4%, respectively) versus the dsodn context (9% vs 21%, respectively). Again, this trend follows those in the literature in which the duplex context favors the less sterically demanding product dgh . The nh3 adducts 5-dsi and 8-dsi provided some interesting context - dependent product distributions . The yield of 5-dsi was greatest for the nucleoside (29%) and decreased by half in all odn contexts studied (14%). In contrast, the yield of 8-dsi was smallest for the nucleoside (11%) and increased by more than 3-fold in the ssodn context (39%), and over half the products observed in the dsodn context were 8-dsi (54%). As previously stated, the lifetime of the electrophilic dg is longer in the duplex context and the superior nucleophilicity of nh3 compared to h2o greatly increased the yield of 8-dsi in dsodn oxidations (scheme 6). Furthermore, the increase in 8-dsi yield from the dsodn oxidations further supports this product resulting from nucleophilic addition of nh3 at c8 leading to 8-dsi, and not the nh3 aminyl radical adding at c8 to yield the same product . These results clearly demonstrate a dependence on the reaction context in formation of dg oxidation products and nh3 adducts . A look at the distribution of diastereomers resulting from oxidation of the odn contexts gave nearly equal amounts of both r and s diastereomers . This observation parallels our previous work looking at dsp diastereomer formation in single - stranded and duplex contexts . Coupled together, these observations support a state of disorder in the duplex at the time of nucleophilic attack at c5 of oxidized dog or 8-amino - dg (likely intermediates leading to the spirocycles) that leads to nearly equal covalent bond formation from both the re and si faces of these electrophiles . Relative product distributions measured when dg was allowed to react with nh3 in the presence of photoexcited riboflavin in various contexts . Reactions were conducted in 75 mm napi buffer (ph 7.4) at 22 c, and 20 mm nh4cl . In the nucleoside studies, 1 mm dg was studied, and in the odn contexts 20 m ssodn or 10 m dsodn was studied . (1) photoactivation of riboflavin (200 m) was achieved with 350 nm light for 3 h in the nucleoside studies and 30 min in the odn studies . These conditions achieved 70% conversion to product in the nucleoside reactions and 50% in the odn studies . It must first be noted that the bis - ammonium adduct could not be quantified due to its instability, but if formed, the adduct is most likely deaminated to either 5-dsi or 8-dsi . Keeping this limitation in mind, the oxidation of dg by riboflavin initially yields dg that rapidly deprotonates to dg in the nucleoside contexts (pka 3.9), dominating the product - forming step of the reaction . In contrast, dg in the dsodn context retains more cationic character because the acidic proton is h - bonded with 2-deoxycytosine in a base pair; therefore, the product - forming steps of the reaction are determined by the radical cation intermediate . As shown in scheme 2, dg could couple with radicals (e.g., o2) to ultimately yield dz, while dg reacts with nucleophiles (e.g., h2o and nh3) at c8, ultimately yielding a spirocyclic product after completion of the four - electron oxidation . Thus, the yield of 8-dsi was anticipated to be greatest in the duplex context, as indeed it is . Furthermore, these results support the original hypothesis by steenken that the duplex context would favor the radical cation nature of one - electron oxidized dg, which is still the focus of many current research efforts . Because of the limitation imposed by the inability to detect the bis - ammonium adduct, or to understand its decomposition pathway, any comparison between the ammonia adduct distributions would be based on poorly defined assumptions; consequently, further discussion of these results is not provided . For the last set of studies, we were inspired by the observation that the a ring of dsp mimics the h - bonding pattern of thymidine, while the a ring of 5-dsi mimics the h - bonding pattern of 2-deoxycytidine (figure 6). On the basis of this observation, polymerase insertion assays opposite dsp vs 5-dsi were conducted to determine if these h - bonding schemes applied during the selection of a nucleotide to insert opposite these spirocycles . Comparison of h - bonding patterns between dsp vs t and 5-dsi vs dc . Site - specific synthesis of dsp or 5-dsi was achieved by synthesizing dog into an odn by solid - phase synthesis and then oxidizing this strand with or without nh4cl present with the one - electron oxidant na2ircl6 to furnish the desired products . Upon ion - exchange hplc purification, the absolute stereochemical assignments for the dsp isomers have been established for the odns, and those for 5-dsi are assigned in this work to be analogous based on the mechanism of deamination discussed above (see figure s7, supporting information, for more details). Standing start polymerase studies were conducted while providing the enzyme only one type of nucleotide per reaction . On the basis of previous studies, insertion of dttp opposite dsp was not observed; therefore, to ensure that only one nucleotide was inserted opposite the lesion, and no extension past the lesions occurs, the sequence had an a placed 5 to the spirocycle (figure 7). Klenow fragment exo- was selected as the polymerase, and after the extension reactions, polyacrylamide gel electrophoresis (page) was conducted to determine the amount of datp, dttp, dgtp, or dctp incorporated opposite the dsp or 5-dsi diastereomers . The polymerase did not select either pyrimidine for insertion opposite the two lesions (figure s7, supporting information); thus, only data for purine insertion opposite the lesions is provided in figure 7 . With respect to insertion opposite the dsp diastereomers, (r)-dsp was observed to give a slight preference for insertion of datp opposite, and (s)-dsp gave nearly equal insertion of datp and dgtp . In comparison to the dsp results, the 5-dsi isomers showed that (s)-5-dsi gives similar amounts of datp and dgtp insertion, while (r)-5-dsi yields a slight preference for datp insertion . Unfortunately, these results do not support the hypothesis that h - bonding in the a ring of the spirocycle is the key parameter for nucleotide selection, but do show that stereochemistry may be important for selecting a base pairing partner by this polymerase . Sequence studied for polymerase dntp insertion studies and the percent dntp insertions opposite the diastereomers of dsp and 5-dsi . (a) sequence for the dsodn construct used in the polymerase insertion assays and (b) comparison of the percent purine dntp insertion opposite the lesions dsp and 5-dsi by the dna polymerase klenow fragment exo- . The present studies mapped the pathways and quantified the products observed when dg was allowed to react with nh4cl in the presence of the photooxidants riboflavin and rose bengal, as well as the one - electron oxidant na2ircl6 . On the basis of the analysis of the products in the nucleoside context, the major products were oxidant dependent . The major product for riboflavin was dz, rose bengal yielded the 5-dsi diastereomers, and na2ircl6 gave the dsp diastereomers as the major products (figure 3). Further analyses determined that 5-dsi and 8-dsi decompose via an acid - catalyzed deamination pathway leading to dsp as an end product (scheme 5). Oxidations of dg in the nucleoside, ss- and dsodn contexts with photochemically activated riboflavin demonstrated three major context effects: (1) the yield of dz was highest in the nucleoside studies and decreased dramatically in dsodn contexts; (2) when nh3 participated as the nucleophile, 5-dsi was obtained in the highest yield in nucleoside studies, while the yield of 8-dsi was highest in dsodn contexts; and (3) when h2o was the nucleophile in nucleoside reactions, dsp presented in the highest yield, while in dsodn contexts dgh was obtained in the highest yield (figure 4). Furthermore, a combination of mapping the decomposition pathways of the dsi compounds to dsp, in which the absolute configuration has been assigned, allowed the determination of the absolute configurations for the diastereomers of 5- and 8-dsi . These studies provide fundamental chemical insight into formation of amine adducts to dg and their stability . Further, these studies insert a cautionary note to researchers who purify their odns using ammonium salts and then conduct oxidation reactions after purification . Ammonia is a better nucleophile and out - competes water for the electrophilic intermediates derived from dg or dog oxidation, resulting in a new mass of dg + 31 or dog + 15, respectively . A similar observation has been highlighted for oxidations occurring in tris buffer that generate tris adducts to dg . Oxidations were conducted with 2-deoxyguanosine (dg) at 1 mm concentration in 75 mm napi buffer (ph 7.4) and 22 c . Reactions with 20 mm nh4cl and without this salt allowed product profile comparisons . The oxidants and specific reaction conditions were achieved as follows: (1) riboflavin oxidations were initiated by adding 200 m riboflavin while exposing the samples to 350 nm light for 3 h. (2) rose bengal oxidations were achieved by adding 100 m rose bengal and exposing these samples to 350 nm for 3 h. the light source for the riboflavin and rose bengal reactions came from a sun lamp that was placed 7 cm above the reaction eppendorf tubes . The tube lids were left open to allow all wavelengths of light to pass into the reaction samples . (3) the na2ircl6 oxidations were initiated by a bolus addition of the oxidizing salt with a final concentration of 10 mm, after a 30 min reaction the samples were quenched with 50 mm edta (ph 8). The reaction products and their distributions were determined by a dual hplc method following a previously reported set of protocols (specific details can be found in the supporting information file). The first rp - hplc run allowed analysis of dog and 8-amino - dg (both observed in <1% yield), while all other products eluted in the void volume of this run . The void volume was collected, dried, and reinjected on a hypercarb hplc column to analyze the diastereomers of dgh, dsp, 5-dsi, and 8-dsi, as well as the product dz, while monitoring their absorbance at 240 nm . To determine product distributions the peak areas were integrated and normalized by each compound s 240 nm (values provided below). Esi - ms, and then each compound was hplc purified for further structural analysis . The dgh diastereomers were previously characterized by nmr, dz was also previously characterized by nmr, and the dsp diastereomers have been characterized by x - ray crystallography . Structural characterization of 5-dsi and 8-dsi by nmr was not conducted because of the instability of these compounds toward deaminating to dsp, and the lack of nonexchangeable protons on the ring of either ammonia adducts makes them very challenging for structural analysis . Furthermore, if deamination of either 5-dsi or 8-dsi to dsp occurs in the nmr tube during analysis, the peaks observed would be similar and challenging to interpret; therefore, the best method for obtaining structural data on the diastereomers of 5-dsi and 8-dsi is via the esi - ms / ms that was conducted (figure 2), and provided satisfactory results to determine their structures . Characterization for each molecule is as follows, the mixture of the dgh diastereomers (not resolvable) gave tr = 6 min; lc esi - ms m / z [m + h] calcd 274.3, found 274.1; hrms (esi - tof) m / z [m + na] calcd for c9h15n5o5na 296.0971, found 296.0979; uv vis 240 = 2400 l molcm. (r)-dsp: tr = 11 min; lc esi - ms m / z [m + h] calcd 300.3, found 300.3; hrms (esi - tof) m / z [m + na] for c10h13n5o6na calcd 322.0764, found 322.0761; esi - ms / ms m / z [m + h] lit . 184, 156, 141, 114, 113, 99, 86, found 184, 156, 141, 114, 113, 99, and 86; uv vis 240 = 3,300 lmolcm; cd (c 1.24 10 m in ddh2o) nm 259 (8.7), 236 (+ 35.1), and 211 (37.4). (s)-dsp: tr = 18 min; lc esi - ms m / z [m + h] calcd 300.3, found 300.3; hrms (esi - tof) m / z [m + na] for c10h13n5o6na calcd 322.0764, found 322.0761; esi - ms / ms m / z [m + h] lit . 184, 156, 141, 114, 113, 99, 86, found 184, 156, 141, 114, 113, 99, and 86; uv vis 240 = 3300 lmol cm; cd (c 1.30 10 m in ddh2o) nm 258 (+ 8.7), 234 (32.9), and 212 (+ 40.5). (r)-5dsi: tr = 9 min; lc esi - ms m / z [m + h] calcd 299.3, found 299.3; hrms (esi - tof) m / z [m + na] for c10h14n6o5na calcd 321.0923, found 321.0920; esi - ms / ms m / z [m + h] found values 183, 166, 140, 123, 113, 96, and 86; uv vis 240 = 3800 lmolcm; cd (c 1.50 10 m in ddh2o) nm 258 (8.2), 242 (+ 20.8), and 216 (+ 61.8). (s)-5dsi: tr = 12 min; lc - esi - ms m / z [m + h] calcd 299.3, found 299.3; hrms (esi - tof) m / z [m + na] for c10h14n6o5na calcd 321.0923, found 321.0916; esi - ms / ms m / z [m + h] found 183, 166, 140, 123, 113, 96, and 86; uv vis 240 = 3800 lmolcm; cd (c 1.45 10 m in ddh2o) nm 258 (+ 8.2), 242 (20.1), and 216 (62.2). (r)-8dsi: tr = 7 min; lc esi - ms m / z [m + h] calcd 299.3, expt 299.3; hrms (esi - tof) m / z [m + na] for c10h14n6o5na calcd 321.0923, found 321.0924; esi - ms / ms m / z [m + h] found 183, 165, 155, 138, 113, 98, and 86; uv vis 240 = 3500 lmolcm; cd (c 1.30 10 m in ddh2o) nm 261 (+ 20.0), 237 (43.2), and 203 (+ 18.6). (s)-8dsi: tr = 14 min; lc esi - ms m / z [m + h] calcd 299.3, expt 299.3; hrms (esi - tof) m / z [m + na] for c10h14n6o5na calcd 321.0923, found 321.0926; esi - ms / ms m / z [m + h] found 183, 165, 155, 138, 113, 98, and 86; uv vis 240 = 3500 lmolcm; cd (c 1.24 10 m in ddh2o) nm 262 (13.7), 239 (+ 46.0), and 201 (33.1). Dz: tr = 27 min; lc esi - ms m / z [m + h] calcd 247.3, found 247.3; hrms (esi - tof) m / z [m + na] for c8h14n4o5na calcd 269.0862, found 269.0870; esi - ms / ms m / z [m + h] lit . 131 and 117, found 131 and 117; uv vis 240 = 1800 lmolcm . The odns were hplc purified using an ion - exchange hplc column, and the purification salts (naoac) were removed by dialysis prior to their oxidation following previously reported methods . The riboflavin oxidations were conducted similarly to those reported for the nucleoside studies, with the following exceptions: the ssodn oxidations were conducted on 20 m samples while the dsodn oxidations were conducted on 10 m samples . In addition, the reaction times were decreased for the odn reactions to 30 min . After the oxidations, the odns were digested with a suite of nucleases and phosphatases to nucleoside samples following a previously established protocol, with the exception that all buffers during the digestion process were comprised of ammonium salts . Next, the digested mixture was analyzed by the hplc method used in the nucleoside studies . The polymerase insertion assays were conducted on duplex odn samples that had site specific incorporation of dsp or 5-dsi in the template strand . The site - specific synthesis was commenced on odns that had a dog phosphoramidite synthesized at the desired site of modification within the sequence 5-cgt tax ggc gca act gga aa-3 where x = dog . The modifications were synthesized by taking 1 nmol of the dog - containing odn and placing it in 100 l of reaction buffer (75 mm napi, ph 7.4) with or without 2 mm nh4cl . The reaction without nh4cl gave the dsp diastereomers and the reaction with nh4cl gave the 5-dsi diastereomers . The individual diastereomers were purified using an ion - exchange hplc column running naoac as the resolving salt and characterized via digestion of the odn to nucleosides followed by hplc analysis (purification details can be found in the supporting information). The primer template duplex for the insertion studies was made by annealing 125 nm primer (5-tt tcc agt tgc gcc-3) with 156 nm lesion - containing template (5-cgt tax ggc gca act gga aa-3 where x = (r)-dsp, (s)-dsp, (r)-5-dsi, or (s)-5-dsi to obtain 100 nm duplex in klenow fragment exo- buffer (50 mm tris, 50 mm nacl, 5 mm mgcl2, 1 mm dtt at ph 8). To a 25 l reaction 20 l of the annealed duplex was added, 1 l of klenow fragment exo- (0.2 units/l), 0.5 l of dntp (500 m stock solution), and 8.5 l of klenow buffer to obtain a 100 nm duplex solution with 10 m dntp and 0.2 u of polymerase . The reaction was incubated at 37 c for 30 min, after which loading dye (95% dmf plus 0.025% bromophenol blue, and 0.025% xylene cylanol) was added to the samples and they were heated at 95 c for 20 min to quench the reaction and denature the dna the denatured samples were loaded on a 20% page gel and electrophoresed for 2 h at 45 w. upon completion of the electrophoresis, the gel was placed in a phosphor screen overnight and imaged by storage phosphor autoradiography.

A review of the literature and an extensive medline search revealed that this is the first case report of the use of guaifenesin to increase sperm motility . He reported an inability to conceive with his wife after 18 months of unprotected intercourse . A semen analysis was performed that included spermatozoa count, liquefaction, morphology, motility, viscosity and volume . Two months after guaifenesin therapy the semen analysis was repeated that demonstrated marked improvement in both total sperm count and motility . Evidence for the effectiveness of guaifenesin is almost entirely anecdotal . Given the mechanism of action of guaifenesin, it is not clear from this case why the patient demonstrated such a large improvement in both sperm count and motility . Additional studies of the effects of guaifenesin on male fertility could yield information of the medication s effect on men with normal or decreased total sperm counts . There are currently anecdotal reports and popular news media stories on the use of guaifenesin, particularly the brand name product robitussin (pfizer, inc ., new york, ny), for use in treating both male and female infertility.14 guaifenesin is an expectorant medication sold over the counter and usually taken by mouth to assist expectoration of phlegm from the airways in acute respiratory tract infections . Its mode of action in treating infertility is not well understood, but it appears to decrease mucus viscosity . A 32-year - old male patient presented to his primary care provider for an infertility evaluation . The patient is a nonsmoker, who consumes little or no alcohol with no known allergies . A recent screening exam for pulmonary tuberculosis was negative and the patient had recently undergone a required military service physical exam . He reported an inability to conceive with his wife after 18 months of unprotected, regular intercourse . As part of a routine infertility evaluation a semen analysis was performed that included spermatozoa count, liquefaction, morphology, motility, viscosity and volume (cpt code 89320). Initial results of the semen analysis demonstrated low sperm count and motility (table 1). This sample, as well as the follow - up sample, were obtained through masturbation and provided to the lab within 30 minutes of collection . The patient s primary care provider offered treatment with guaifenesin 600 mg extended release tablets twice daily . The repeat semen analysis demonstrated marked improvement in both total sperm count and motility (table 1). The patient made no other significant lifestyle changes during the treatment course with guaifenesin . At the time of writing this case report describes the semen analysis laboratory results in a male patient who was given guaifenesin . Guaifenesin is a mucolytic agent usually taken orally to assist the expectoration of phlegm from the airways in acute respiratory tract infections . Scientific evidence for the effectiveness of guaifenesin is almost entirely anecdotal; a review of medical literature revealed very limited data on use of guaifenesin for infertility.5,6 there appeared to be some improvement in a small study without controls of female infertility related to hostile cervical mucus.5 check regards guaifenesin as the simplest but least effective method of improving cervical mucus.7 given the proposed mechanism of action of guaifenesin, it is not clear from this case why the patient demonstrated such a large improvement in both sperm count and motility . Additional study of the effects of guaifenesin on male fertility suggests the need to conduct a more rigorous placebo - controlled clinical trial that could yield information of the medication s effects on men with normal or decreased total sperm counts.

Agriculture has been one of the primary economic avenues in the philippines contributing to about 20% to the gross domestic product (gdp). Crops comprise about 47.56% of the total agricultural sector and have contributed to about 510 billion pesos (p510b) to the country's national income . Benguet is a province in the northern portion of the philippines belonging to the cordillera administrative region . There are about 27.5 thousand farms covering 30 thousand hectares of agricultural land in benguet . It is also the largest producer of vegetables and fruits, supplying the capital cities in the philippines . The province is known as the salad bowl of the philippines as its major crops are tubers, roots, bulbs, leafy vegetables, stems, and flowers . In 2005, benguet was the top producer of brocolli and carrots producing about 1.2 thousand and 13.7 metric tons contributing to 87.4% and 81.4%, respectively, to the national output . However, growing vegetables is considered a risk occupation in some areas in developing countries . Soogarun et al . In 2003 found significantly low / abnormal mean blood cholinesterase levels among vegetable growers in thailand . Health impacts of pesticide misuse on the other hand greatly affect the farming communities in the philippines questioning the economic advantages of its use . Many researchers have correlated the extent of direct and indirect pesticide exposure and health hazards such as increased mortality, dermal contamination, depression in cholinesterase level, fetal abnormalities, and spontaneous abortion among pregnant women [36]. It is a discouraging fact though, that with knowledge of health risks, many filipino families still perceive that crop yield outweighs the health risks associated with pesticide use . Pesticide poisoning is one of the most prevalent health problems in the philippines . In a study by the department of health (doh) from 19911995, organophosphates accounted for the highest number of poisoning cases while organochlorines caused the most number of deaths . Cheng in 1994 studied 2000 benguet vegetable farmers and found that the most common complaints were allergic reactions both in the skin and the eyes, abdominal pain, dizziness, chest pain, headache, and nose bleed . Meanwhile, a study on pesticide poisoning in selected hospitals in four philippines regions in 2001 found that cases of acute poisoning were more prevalent than chronic cases . This study aimed to identify the pesticide exposure and risk factors among vegetable farmers . The data can be used as baseline data on the vegetable industry in the philippines . This was a cross - sectional study to investigate the prevalence of pesticide exposure and its risk factors . Target population consisted of vegetable farmers in the largest vegetable producing community in the philippines . The inclusion criteria were farmers living in the community for at least one year from the time of interview, and practicing farmers who own or work a farm in the community . Those who were involved in organic farming and the migrant farmers who have been in the area for less than one year were excluded . There were 211 respondents from the identified municipalities selected as the study population using cluster sampling . The sampling size calculated with p = .05 was 211 vegetable farmers and 37 farms . Data gathering was done using the following: (1) questionnaire structured personal interview with farm workers / farmers was done by research assistants who were trained prior to the data collection . Details included personal information, health history, pesticide usage, work practices, work conditions, risk factors associated with pesticide exposure, and health data; (2) exposure assessment monitoring on work conditions, work practices, and pesticide concentration; (3) work analysis in each farm was also done to validate work practices related to pesticide preparation and application . Recall bias was dealt with by confining the health data questionnaire to the last one year from the time of interview . The health data were also collected by medical doctors who simultaneously conducted physical assessment of the farmers . This project study was collaborated with the local agencies coordinating with farmers in the vegetable industry in benguet . Ethical clearance was given by the research, information and dissemination office of the proponent's institute . The study included 127 males (60.2%) and 84 females (39.8%) with ages ranging from 16 to 72 (mean = 45 12) showing a relatively adult population . Seventy one percent (71%) were married and majority were working as agricultural workers (82%), and the remaining were pesticide applicators, mixers, and loaders (18%). The respondents were living in their present address for an average of 34.76 years (sd = 16.72) with a mean distance of 3163 meters (sd = 36539.13) from the vegetable plantation or farm . Few farmers reported history of smoking (16.2%), and 7% claimed they smoked and 2% had a history of chewing tobacco . The average number of cigarettes and tobacco consumed in a week were 12 sticks and <1 tobacco, respectively . The farmers used pesticides in their farms in an average of 1.9 days per week . The mean total application time was 3.47 hours (mean = 3.47 2.09). The mean amount of pesticide used in an application was 21.35 l per application (mean 21.35 48.17). The farmers also reported that in an average year, there were 2.3 (mean = 2.3 0.53) cropping seasons with a mean of 3.84 (mean = 2.3 0.53) months per cropping season (table 1). 44.5% reported that they wiped their sweat with a contaminated piece of fabric, 41.7% re - entered recently sprayed area, 37.4% had exposure because of damaged backpack sprayer, and 31.8% were exposed when they sprayed against the wind (table 2). However, further analysis shows that they did not frequently use such equipment nor had adequate gear to fully protect themselves . The same pattern was seen among all kinds of personal protective equipment (ppe) with the exception of boots which was frequently used by 77.5% of farmers . 94% said that they have worked with or used pesticides in their lifetime, and 16.4% from this population used pesticides in their own households . The vegetables commonly grown in the area were potatoes (67.4%), cabbage (63.7%), and carrots (36.8%). Majority (87%) the most common route of pesticide entry in the study was respiratory (68.9%) followed by dermal and ocular entry (60.5% and 38%, resp . ). Sumicidine was the most commonly used pyrethroid which contains fenvalerate as its active ingredient . Table 3 shows that 37% and almost 14.7% of the study population used pesticides with active ingredients of fenvalerate and cypermethrin, respectively . Although pyrethroid was the most frequently used pesticide, it is organophosphate that consisted the largest amount of exposure among farmers at 210.02 liters, followed by pyrethorid at 151.4 liters, and carbamates at 32.16 liters for the entire one year . The pesticide exposure of the farmers measured in table 4 as dependent variable was related to the amount of pesticide used in liters, frequency of use and duration of use . All the independent variables, except amount and years of pesticide use were categorical variables . Those who used more pesticides over a longer period of time had higher total pesticide exposure . Furthermore, a farmer who was a pesticide applicator, mixer, loader and who wiped sweat with contaminated piece of fabric, and who had not been given instructions through training association was at risk of having higher pesticide exposure . Seventy four percent (74%) of the respondents became ill because of work for the last 12 months preceding the study . The most common symptoms were headache (64.1%), muscle pain (61.1%), cough (45.5%), weakness (42.4%), eye pain (39.9%), chest pain (37.4%), and eye redness (33.8%). A subsequent study is recommended to focus on adverse health effects of these farmers and association with certain risk factors . The results of this study identified pesticide exposure and farming practices of farmers in the largest vegetable producing area in the philippines . This has also been documented in other countries . In the study of coble et al . In 2005, and thompson et al . In 2003, unsafe practices like re - entry of recently spayed area, use of damaged backpack sprayer and wiping sweat with a contaminated piece of fabric were identified in this study . Re - entering a recently sprayed area, as mentioned in the study of tielemans et al . In 1999 is an important determinant of dermal exposure to specific chemicals such as captan and tolylfluanid . A pesticide's formulation is a significant factor for human exposure, with greater risks present among aqueous and emulsifiable concentrates because it impairs the protective function of chemically protective gloves . According to wolfe in 1993, pesticides may react through chemical and biotic processes . However, pesticides may undergo activation processes unexpectedly and may be broken down to equally or more potent and mobile toxic compounds posing a greater threat to nontarget organisms . It is then advised to limit or decrease the frequency or duration of staying in the contaminated crops right after pesticide application . Organophosphates, carbamates and pyrethroid pesticides were the most commonly used type of pesticides among the farmers in this study . The same was seen in the study of clarke et al . In 1997 in ghanna where organophosphates consisted of the most commonly used pesticides followed by carbamates and organochlorines . The same trend was seen among farmers in sri lanka and in brazil [15, 16]. The farmers in this study used pesticides in their farms with a mean application time of 3.47 hours (mean = 3.47 2.09). The mean amount of pesticide used in an application was 21.35 liters per application (mean 21.35 48.17). The number of spray operations per week has been proven to have significant association with the likelihood of experiencing neurobehavioral, respiratory, or intestinal symptoms in a study among indonesian farmers . In a study among north carolina growers and agents, it was found that the study population perceived that once the pesticide is diluted and reentry intervals are observed, the risk it poses becomes diminished . Sumicidine, which was the most commonly used pesticides among the benguet farmers contains fenvalerate . Fenvalerate induces numbness, itching, tingling, and burning sensations in exposed workers that developed after a latent period of approximately 30 minutes, peaked by 8 hours, and disappeared within 24 hours . Additional data among chinese workers demonstrated that fenvalerate decreased the semen quality of occupational workers . On the other hand, other active ingredients like cypermethrin, mancozeb, and skin sensations were reported to occur among field workers and usually lasted only for a few hours and did not persist for more than one day after exposure to cypermethrin . For mancozeb, prolonged low - level exposure to mancozeb affected several aspects of immune functioning and moderate association existed between mancozeb and neural tube defects . Most often, gloves were the most commonly used personal protective equipment because the hands were the most exposed areas [23, 24]. Many circumstances contributed to nonadherence to proper use of ppe like extreme heat during pesticide application, uncomfortable to use, few resources to afford new ppe, peer - related factors, and increasing age [18, 2527]. The study also showed certain risk factors associated with pesticide exposure such as re - entering recently sprayed area, spraying against the wind, use of damaged backpack sprayer, spills on the back, spills while mixing pesticides, among others . Aside from direct pesticide use, the different agricultural tasks mentioned above may also contribute as risk factors to pesticide exposure . Re - entering a recently sprayed area, as mentioned in the study of tielemans et al . In 1999 is an important determinant of dermal exposure to specific chemicals such as captan and tolylfluanid . There is evidence that weight loss could be a possible health effect of chronic pesticide poisoning . Decreased body mean mass accompanied by reduced cholinesterase activities among seven farm workers was documented . Also, kackar et al . In 1999 found that when rats were administered orally with mancozeb (ethylenebisdithiocarbamate), dose - dependent signs of poisoning, weight loss, and mortality developed . Senthilselvan et al . Found a significant association between carbamate exposure and prevalence of asthma among those non - asthmatic farmers and lower mean lung function variables among those with asthma . This study has shown the pesticide exposure of farmers in the largest vegetable producing area in the philippines . It is vital that a sequential exposure assessment be done in order to come up with a correlation study between pesticide exposure and health problems . The study showed that pesticide use is prevalent among farmers in benguet which is the largest vegetable producer in the philippines . It is also suggested that chronic effects of pesticide cited in certain studies [31, 32] such as carcinogenic effects, poor reproductive outcomes, neurologic and respiratory disorders, impairments of the immune system and birth defects should also be investigated in future studies . This manuscript adds to existing literature on pesticide exposure in the philippines which are so far mainly descriptive in nature . This paper also identifies risk factors such as work practices and designs of containers / sprayers that may increase pesticide exposure among farmers . This also calls for a local level policy research for program intervention among vegetable farmers using pesticides.

Evans, using a mouse mutant for the lim homeodomain transcription factor (islet1, isl1), demonstrated that an isl1 positive (isl1 +) population is localised in the secondary heart field (shf), contributing to the outflow tract, right ventricle, atria and (to a small extent) the left ventricle . Isl1 is considered a marker of shf cells that contribute to the venous and arterial poles of the cardiac tube, while the other cells from the shf contribute only to the arterial pole . The shf or anterior heat field (ahf) was identified as a splanchnic mesoderm underlying the caudal pharynx, which provides myocardium to the outflow tract during tube looping . In 2005, the same research group also identified isl1+/c - kit negative (c - kit-)/stem cell antigen-1 negative (sca-1-) progenitors in the postnatal heart, demonstrating that these cells persist after birth and that these organ - specific progenitor cells may be isolated, purified, expanded and differentiated into mature cardiac myocytes for future cardiac applications . Our research group identified isl1 + cells during gestation until the postnatal age, and demonstrated the contemporary expression of isl1 and c - kit in the interstitial cells in the ventricular myocardium in human foetal and postnatal hearts . This paper demonstrated, by confocal analysis, that all isl1 + cells were also c - kit+, while c - kit+ cells were not necessarily isl1 +, as if isl1 + cells were a subpopulation of c - kit+ cells . It has been supposed that there may be a primordial cardiovascular precursor in the shf that expresses the three markers isl1, nkx2.5 and fetal liver kinase 1 (flk1) and can generate three lineages: cardiac muscle, smooth muscle and endothelium . The shf cooperates with the first heart field (fhf) and the cardiac neural crest cells during the development of the vertebrate heart . The fhf lineage forms the early cardiac tube and generates the left ventricle, while the shf contributes additional cells to the maturing heart and, along with the cardiac neural crest cells, generates the outflow tract vessels and valves . The presence of cardiovascular progenitor cells positive for isl1 and/or nkx2.5 in the shf has been well confirmed by a recent work published in science . The authors suggested that there are two distinct populations, one for the shf and the other for the fhf; the first population is clearly positive for isl1 and expresses mirna 199a / b, while the second has no distinct markers and expresses mirna 200a / b . Progenitor cells of the shf depend on the cardiac specific transcription factors isl1 and nkx2.5, which play a key role at an early stage of cardiac progenitor formation . Their expression is regulated by wnt / beta - catenin signalling, which in turn also regulates bmp4 signalling . This last signalling pathway activates gata4 and srf . Despite the key role of isl1, this transcription factor is not considered an indubitable marker of this subpopulation of shf progenitor cells . It has been demonstrated very recently that isl1 labels both shf precursors and some cardiac neural crest cells and that mef2c is a more efficient marker of the shf progenitor cells in labelling experiments . This paper indirectly suggests both a review of past literature in view of these new data and using a different marker in studies of the shf progenitor cells . Moreover, considering heart development in different species, such as chicken, mouse and xenopus, it appears that isl1 is expressed in a common cardiac cell population that splits into fhf and shf lineages during heart development and that this common progenitor also contributes cells to the cardiovascular system . Anversa s research group described rat cardiac stem cells for the first time, many research groups have claimed to have discovered new and important cardiac progenitor / stem cells in the adult heart .. since 2005, the scientific community talked about adult c - kit+ and/or sca-1 + and/or mdr-1 + cells, and embryonic isl1 + cells . These two populations have always been considered different entities and are described separately in many research papers and reviews, although expression of the isl1 transcription factor by cardiac precursor cells has also been reported in adult hearts . Di nardo, described for the first time the concomitant expression of the three markers, i.e., c - kit, sca-1 and isl1, in the same cardiac precursor cell . Prof . Di nardo s research group described the concomitant expression of the isl1, c - kit and sca-1 markers in adult mouse cardiac progenitor cells in 2008, while in 2009 our research group confirmed the expression of the three markers in adult rat cpcs . In a systematic work published in 2011 about the identification of isl1 + cells in the mouse heart from postnatal day 1 to young adulthood in different strains, the authors found clusters of positive cells in the cardiac ganglia of the studied strains, while found very a few clusters of isl1 + cardiac precursors only in 129svj or balb / c strains and in animals not older than 4 months . Recently genead et al . Demonstrated the contemporary expression of c - kit and isl1 markers in rat adult hearts in normal, pregnant and infarcted individuals . They reported the expression of both markers in the entire heart and in the right ventricle, the left ventricle, the outflow tract and the peri - infarct and peri - ischemia regions . Unfortunately, the study was based on real - time pcr analysis, and whenever immunocytochemistry was shown, no double staining for both markers had been performed . Other evidence supports the hypothesis that isl1 + cells are not a different population from sca-1 + cpcs . The contemporary expression of sca-1 and isl1 has been described both in a subpopulation of sca-1+/c - kit- cells identified and isolated from adult mouse hearts and in sca-1 + cardiosphere - derived cells obtained from cardiac explants from normal, sham - operated or post - myocardial infarct hearts . In this study, only sca-1+/cd45- cells were also positive for isl1 and increased in number only after an acute myocardial infarct . Considering that other authors identified isl1 + cells in adult murine and rat hearts, weinberger et al . Used heterozygous isl1-lacz mice, a more sensitive genetic approach, to investigate the presence and localisation of these precursor cells in 30 animals at different time points after birth (10 weeks to 18 months). They found four different populations of isl1 + cells: i) clusters of isl1 + neurons were found in the cardiac parasympathetic ganglia of the posterior side of the heart and in the nervous plexus surrounding the pulmonary veins; ii) clusters of isl1 + smooth muscle cells were found in the muscular layer above the aortic and pulmonary valve in the proximal part of the aorta and the trunk of the pulmonary artery (only a few positive cells were present in the aortic valve leaflets); iii) clusters of isl1 + cardiomyocytes were found in the left ventricular outflow tract region; iv) clusters of isl1 + sinoatrial node (san) cells were found in the muscular wall between right atrium and vena cava superior . These results support the hypothesis that in the adult heart many cell populations may derive from isl1 + embryonic precursors, as smooth muscle cells, parasympathetic neurons, san cells, but also that, even if reduced in number, isl1 + cardiomyogenic progenitors, may be present in the adult myocardium . Many papers have been published on cpcs, and many research groups claimed to have discovered different populations of progenitor cells . It is strange to think that the cardiac tissue possesses a larger number of stem / progenitor cells compared to the other tissues found in our body, and it is hopeful that cpcs may vary in number with external stimuli or with age as it happens for other adult stem cells . The hypothesis that the myocardium is a non - regenerative tissue is not valid anymore, and much evidence supports the new idea that there is at least one cardiac progenitor cell . Isl1 + and c - kit+/sca-1 + cells were considered to be two different populations until 2012, when prof . Sussmann, citing our recent paper published in the european journal of histochemistry in an editorial in circulation research, suggested considering the possibility of studying a population of cells positive for both isl1 and c - kit instead of choosing to study only one side of the coin . In the review and in the research paper published in 2009, we reported the concomitant expression of isl1, c - kit and sca-1 in a population of cpcs isolated without any sorting or selection and suggested for the first time that there could be a unique population of cells with several subpopulations and that the several markers identified could be only the effect of different cultivation conditions in the different laboratories . Other research groups later described the co - expression of isl1, sca-1 and/or c - kit and we further demonstrated that, in samples from foetal and postnatal human hearts, there was also an isl1 + subpopulation of c - kit+ cells . Anversa among the authors, on a study of the presence of c - kit+ cardiac stem cells in embryonic, foetal and neonatal mouse hearts . They demonstrated that these cells can form cardiomyocytes and coronary vessels and, as we have demonstrated in our paper, they are more abundant than isl1 + cells . Moreover, they stated that isl1 + cells are present only in the outflow tract, atria and part of the right ventricle and that they are a population of committed cells derived from an immature primitive cell . We can say that there is strong evidence suggesting that c - kit, sca-1 and isl1 may be markers of subpopulations of cpcs that are present in the embryonic, foetal and neonatal heart and persist from after birth until adulthood . The identification of the real immature cardiac precursor expressing the four cell markers (c - kit, sca-1, mdr-1 and isl1) and above all the identification of a unique isolation protocol is very important in the clinical practice . This immature common cardiac precursor may represent the perfect candidate for cardiac tissue engineering and, hopefully, may be driven pharmacologically to one phenotype or the other . To identify a standardised protocol is extremely important to go further in the studies on cardiac tissue regeneration, and common guidelines are needed in cardiac stem cell therapy . The isolation of stem cells by cell sorting or immunobead selection using surface markers such as c - kit, sca-1 or mdr-1 is important to identify a standardised protocol of isolation, however isl1 may be useful in basic research to follow the fate of cpcs in heart development and myocardium differentiation . Moreover, it is extremely important to understand which is the predominant precursor population in the different stages of life and which population should be used in a paediatric patient instead of an adult one . If fate of immature cardiac precursors may be determined pharmacologically, the possibility to regenerate san cells is very important in curing arrhythmia s both in patients with ischemia and malformations . The identification of the different subpopulations in the developing heart may be a fundamental step in identifying the optimal cell line for cell - based therapies.

Primary diffuse leptomeningeal gliomatosis (pdlg) is a rare neoplasm with a short survival time of a few months . We report on a 53-year - old male patient who presented with epileptic seizures, gait disturbance, paraparesis and sensory deficits in the dermatomes t8 - 10 . Magnetic resonance imaging (mri) revealing numerous spinal and cranial gadolinium - enhancing nodules in the meninges and histopathology led us to diagnose primary diffuse leptomeningeal gliomatosis with who grade iii astrocytic cells . Consecutively, the patient underwent craniospinal radiotherapy (30 gy) and 11 sequential cycles of temozolomide . Thirteen months later, spinal mri revealed tumor progression . Second - line chemotherapy with 5 cycles of irinotecan and bevacizumab did not prevent further clinical deterioration . The patient died twenty - two months after diagnosis, being the longest survival time described thus far with respect to pdlg consisting of astrocytic tumor cells . Radiochemotherapy including temozolomide, as established standard therapy for brain malignant astrocytomas, might be valid as a basic therapeutic strategy for this pdlg subtype . Primary diffuse leptomeningeal gliomatosis (pdlg) is diagnosed when a glioma is located in the subarachnoid space, while intraparenchymal tumor lesions are absent . Pdlg must be distinguished from secondary meningeal gliomatosis resulting from a primary gliomatous cns tumor . Pdlg, especially when caused by malignant astrocytic cells, is associated with very poor survival . We describe a patient with pdlg consisting of malignant astrocytic cells who underwent combined radio- and chemotherapy leading to the longest survival time described in the literature thus far . A 53-year - old man was referred to the dept . Of neurology due to a generalized epileptic seizure and five months later he presented with bilateral sensory deficits at dermatome levels t8 - 10, paraparesis and gait disturbance . Cerebrospinal fluid (csf) analysis revealed a high cell count (300/3 cells/l; reference range: 5/3 cells/l) with atypical cells that could not be further characterized . T1-weighted images (t1-wi) revealed spinal and cranial gd - enhancing nodules in the leptomeninges (figures 1a - e) and on t2-weighted images (t2-wi) an intramedullary edema (figure 1c). Note gd - enhanced supra- and in - fratentorial meningeal thickenings of the brain in sagittal t1-w image (a). Prior to ra - diochemotherapy, t1-wi reveals lep - tomeningeal gd - enhancement (b) and t2-wi shows edema of the myelon on levels c7, t1 - 2 and t5 - 6 due to minor tumor extensions in the adjacent spinal cord (c). Following radiochemotherapy, t1-wi displays reduced leptomeningeal gd - enhancement (d) while t2-wi shows less myelon spinal edema on those levels (e). Three weeks later, intradural biopsy from a gd - enhancing nodule at l2 - 3 level revealed fibrously - thickened meninges infiltrated by malignant astrocytic tumor cells (figures 2a - c). The tumor was categorized as primary diffuse leptomeningeal gliomatosis (pdlg) confirmed by the reference center for brain tumors in dsseldorf, germany . Intradural lumbar biopsy displays fibrously - thickened lep - tomeninges infiltrated by pleomorphic neoplastic astrocytic cells (a). The astrocytic tumor cells reveal intensive intracytoplasmic staining with anti - glial fibrillary astrocytic protein . Ki67 (mib-1) staining reveals a high proliferation index of up to 20% (c). Prior to radiotherapy of the craniospinal axis (4 2.5 gy / week, total dose: 30 gy), the patient underwent 3 cycles of temozolomide (tmz) (cycle 1: 150 - 200 mg / mtzm, d1 - 5, q28d). On re - staging, spinal t1-weighted images showed tumor - lesion regression on l5 and spinal t2-wi images demonstrated spinal - edema regression (compare figures 1c - d with figures 1a - b). Three weeks after the completion of radiotherapy the patient received eight additional cycles of tmz (200 mg / mtzm, d1 - 5, q28d), during which meningeal thickening of the brain regressed . However, spinal mri thirteen months after starting tmz therapy revealed meningeal tumor progression at the lesions on spinal levels c1 - 2, c7-t2 and t5 - 8 . Five cycles of second - line chemotherapy with irinotecan and bevacizumab failed to halt further clinical deterioration . The patient died twenty - two months after the diagnosis of pdlg and exhibited until dead an encouraging karnovsky performance status of 60% . The diagnosis of pdlg is usually established by autopsy but rarely diagnosed prior to death . Pdlg of the oligodendroglial and that of the well - differentiated astrocytic tumor type are associated with a considerably longer median survival time than that of the malignant astrocytic tumor type . Pdlg was diagnosed in the aforementioned case, as we observed on histopathology leptomeningeally - encapsulated malignant astrocytic cells without primary attachment to the spinal cord or brain parenchyma and gd - enhancing leptomeningeal thickening at the base of the brain and spinal level in mri . Various treatment modalities were used for 14 patients suffering from pdlg with malignant astrocytic cells reported in the literature so far (cf . Table 1), demonstrating the lack of a standardized treatment regimen of pdlg with malignant astrocytic cells . Although the number of cases is small, the data on their survival times (cf . Table 2) might suggest that radiotherapy and temozolomide (tmz) as established treatment for newly diagnosed recurrent anaplastic astrocytomas also seem to be valid for pdlg with malignant astrocytic cells . This concept is supported by the observation that radiotherapy alone can prolong the median survival time of five months for patients without any specific therapy to a median survival of 12 months and that integration of tmz in the chemotherapy alone may lead to a prolonged median survival of 15 months . The importance of tmz in the chemotherapy treatment of pdlg is supported by the observation that the median survival time fell to 3 months when the integration of tmz was omitted . Clinico - pathological characteristics and treatment of adult patients suffering from pdlg with malignant astrocytic cells including our case 5-fu = 5-fluouracil, adr = adriamycin, auc 5 = carboplatine, bcnu = carmustine, brs = brainstem, bvz = bevacizumab, c = cycle, ca = cytarabine, ccnu = lomustine, cr = cranial, cyc = cyclophosphamide, ddp = cisplatin, eto = etoposide, inn = topotecan, int = irinotecan, irp = raised intracranial pressure, it = intrathecally, mcnu = ramustine, mtx = methotrexate, sc = spinal cord, tmz = temozolomide, top = thiotepa, vincristine, prednison summary of the literature: pdlg with malignant astrocytic cells the prolonged survival of our patient may be due to the addition of tmz to radiation therapy as it resulted in an extent of the median survival time for high grade glioma patients . In addition, the hypofractionated radiotherapy regimen used is supposed to be more effective than conventional fractionated irradiation . Additionally, the patient's good karnofsky performance status may have contributed to our patient's prolonged survival . In conclusion, we report on partial regression and long survival in a patient with pdlg of the malignant astrocytic type following hypofractionated radiotherapy and tmz . An immediate radiochemotherapy seems crucial for a prolonged survival of pdlg patients in good general condition . The authors disclose no or potential conflict of interest including any financial, personal or other relationships with other people or organizations within the three years of beginning the submitted work that could inappropriately influence or be perceived to influence their work.

In past years, numerous studies have described the role of microalbuminuria (mau) as a predictor of cardiovascular disease (cvd) and death among subjects with type 2 diabetes (t2d).13 mau is one of the earliest clinical signs for diabetic nephropathy and a significant risk factor for progression to proteinuria.1 additionally, hypertensive t2d individuals with mau have an increased risk of developing end - stage renal disease (esrd).4 risk factors known to be associated with cvd and diabetic nephropathy are high blood pressure (bp) and elevated glycosylated hemoglobin (a1c).5,6 achieving adequate bp and glycemic control (gc) plays an essential role in preventing renal and cvd events in individuals with t2d . A current secondary analysis from the hispanic health and nutritional examination survey (hhanes)7 indicated that cuban americans have higher serum cholesterol and systolic bp than puerto ricans and mexican americans . Furthermore, compared with other hispanic subgroups, cuban americans have the highest proportion of hypertension (htn) and mean serum creatinine levels.8 smith and barnett9 examined the national center for health statistics (nchs) from 1996 to 1997 and concluded that cuban americans 35 years of age and older have the highest percentage of diabetes - related deaths compared with other hispanics . Although previous studies have shown significant differences and diversity within hispanics, further studies conducted in the cuban american population are scarce . Over the past decade, the prevalence of t2d has increased, especially among cuban americans who have a higher incidence of diabetes (8.2%) compared with 6.6% of non - hispanic whites.10 the high incidence of t2d combined with an increased risk for developing diabetes complications warrants further examination of the cuban american population . Screening for mau can detect individuals at risk for renal dysfunction and cvd events and possibly reduce the burden associated with diabetes complications . Therefore, the purpose of this study was to investigate to what degree the coexistence of htn and poor gc influences the likelihood of having mau among cuban americans with t2d . It was hypothesized that individuals with t2d, htn, and poor gc will have an increased likelihood to test positive for mau . It was further hypothesized that this association will be stronger after controlling for confounding variables . This was a cross - sectional study conducted in cuban americans with and without t2d . Data from a complete sample set of cuban americans with and without t2d were used in the present study . Recruitment of participants was conducted in alternate phases of potential subjects with and without t2d, age matching subjects by age groups . During a 1-year period, approximately 10,000 letters outlining the study were mailed to subjects aged 30 years or older with and without diabetes . Letters were sent in english and spanish and included an invitation flyer to which interested participants could respond . The participants were initially recruited by random selection (every tenth address) from a randomly generated mailing list . This company provided a mailing list of cuban americans identified as with and without t2d from miami - dade and broward counties, florida . Three percent (n = 300) of the letters were returned due to unknown addresses . From interested participants were initially interviewed on the phone, at which time the study purpose was explained and the age and gender of the responders were determined . To ascertain t2d status, only 18 subjects did not qualify for the study for not being cuban americans (n = 2), age younger than 30 years (n = 9), and having other chronic illnesses (n = 7). If a subject was determined to be eligible, then their participation was requested at the human nutrition laboratory at florida international university (fiu). Participants were instructed to refrain from smoking, consuming any food and beverages except water, and doing any unusual exercise for at least 8 hours prior to their blood collection . The purpose and protocol of the study were explained to the subjects, and their written consent, either in spanish or english, was obtained prior to the commencement of the study . Seven participants reported not having diabetes but were reclassified because their lab results classified them as having t2d according to american diabetes association (ada) standards . For the data analysis, subjects with caloric intakes> 5000 kcals (n = 2) and missing a1c levels (n = 2) were excluded . For two participants, we were unable to perform a1c analysis . In total, we included only the data from subjects with t2d (n = 179) who were aged 30 years and older . A sociodemographic questionnaire was given to each participant to complete, which included questions related to age, gender, smoking status, medications for diabetes, htn, and cholesterol . Height and weight were measured using a seca balance scale (seca corp, columbia, md, usa). Body mass index (bmi) was calculated as weight in kg / height in m. bp was measured twice then averaged in participants in a sitting position after a 15-minute rest using a random zero sphygmomanometer (tycos 5090 - 02 welch allyn pocket aneroid sphygmomanometer, arden, nc, usa) and a stethoscope (littmann cardiology, 3 m, st paul, mn, usa). Htn was defined as follows: systolic bp 140 mm hg systolic or diastolic bp 90 mm hg or using antihypertensive treatment.11 dietary intake was measured using a validated semiquantitative food frequency questionnaire (ffq) developed by willett et al.12 this ffq has also been validated by nath and huffman13 exclusively for the cuban american population . Participants self - reported average consumption of specified amounts of various foods over the past year and chose from frequency responses ranging from never to six or more servings per day . In addition to food items, the ffq included questions about type and duration of vitamin / mineral supplement use, alcohol consumption, and specific details about fat, salt, and sugar used in cooking and as condiments . Macro- and micronutrient intake was calculated by multiplying frequency of consumption by the nutrient value of the food item obtained from the harvard university food composition database (boston, ma, usa). Venous blood (20 ml) was collected from each subject after an overnight fast (at least 8 hours) by a certified phlebotomist using standard laboratory techniques . Blood samples were collected into a vacutainer serum separator tube (sst) (becton, dickinson and company, franklin lakes, nj) for analysis of lipids and another tube containing ethylenediamine tetraacetic acid to analyze a1c . After coagulation was completed (3045 minutes), the sst was centrifuged at 2500 rpm for 30 minutes . Lipid panel was assayed by enzymatic methods, and a1c percentages were measured from whole blood samples with the roche tina - quant method by laboratory corporation of america, miami, fl, usa (labcorp). Poor gc was defined according to the ada standards (a1c> 7%).14 fresh, single - voided, first morning urine samples were collected from each participant to determine mau by a semi - quantitative assay (immunodip, diagnostic chemicals limited, oxford, ct, usa). Immunodip urinary albumin test uses a monoclonal antibody against human serum albumin to detect mau . The study conducted by davidson et al15 was designed to evaluate the clinical performance of the immunodip dipstick compared with a reference measure recommended by the ada for detecting mau (albumin: creatinine ratio <30 ug / mg [negative];> 30 ug / mg [positive]) determined by laboratory techniques (quest hitachi 717 autoanalyzer). Additionally, results from the immunodip were compared with quantitatively measured albumin concentrations as a secondary outcome . Urinary albumin concentrations were considered <18 mg / l (negative) and> 18 mg / l (positive). Screening for mau with immunodip exhibited a sensitivity of 96% and specificity of 80% when compared with albumin: creatinine ratio> 30 ug / mg . When immunodip was examined against quantitatively measured albumin concentrations, the dipstick yielded a sensitivity of 95% and specificity of 94% . Recommendations from the national academy of clinical biochemistry (nacb) for the diagnosis and management of diabetes indicated that a useful semiquantitative screening test for mau should have a sensitivity> 95%.16 the immunodip dipstick fulfilled the requirements from the nacb as a screening tool to detect mau . In our study, this cut - off value was established by the manufacturer (immunodip, diagnostic chemicals ltd) and corresponded to albumin: creatinine ratio> 30 ug / mg values for mau detection.15 all analyses were performed using spss version 17 (spss inc ., chicago, il, usa). T - tests and chi - square tests were performed to compare means and proportion differences between subjects with and without mau . Unadjusted odds ratios and logistic regression analysis were conducted to investigate the extent to which htn and gc are associated with an increased likelihood of having mau . Controlled variables included in the logistic regression analysis were age, gender, bmi, known duration of diabetes, total cholesterol levels, diabetes and cholesterol medications, smoking, total kcal and protein intake, and intake of potassium, phosphorous, and sodium . This was a cross - sectional study conducted in cuban americans with and without t2d . Data from a complete sample set of cuban americans with and without t2d were used in the present study . Recruitment of participants was conducted in alternate phases of potential subjects with and without t2d, age matching subjects by age groups . During a 1-year period, approximately 10,000 letters outlining the study were mailed to subjects aged 30 years or older with and without diabetes . Letters were sent in english and spanish and included an invitation flyer to which interested participants could respond . The participants were initially recruited by random selection (every tenth address) from a randomly generated mailing list . This company provided a mailing list of cuban americans identified as with and without t2d from miami - dade and broward counties, florida . Three percent (n = 300) of the letters were returned due to unknown addresses . From the remaining delivered mail, 4% (n = 388) responded . Interested participants were initially interviewed on the phone, at which time the study purpose was explained and the age and gender of the responders were determined . To ascertain t2d status, only 18 subjects did not qualify for the study for not being cuban americans (n = 2), age younger than 30 years (n = 9), and having other chronic illnesses (n = 7). If a subject was determined to be eligible, then their participation was requested at the human nutrition laboratory at florida international university (fiu). Participants were instructed to refrain from smoking, consuming any food and beverages except water, and doing any unusual exercise for at least 8 hours prior to their blood collection . The purpose and protocol of the study were explained to the subjects, and their written consent, either in spanish or english, was obtained prior to the commencement of the study . Seven participants reported not having diabetes but were reclassified because their lab results classified them as having t2d according to american diabetes association (ada) standards . For the data analysis, subjects with caloric intakes> 5000 kcals (n = 2) and missing a1c levels (n = 2) were excluded . For two participants, in total, we included only the data from subjects with t2d (n = 179) who were aged 30 years and older . A sociodemographic questionnaire was given to each participant to complete, which included questions related to age, gender, smoking status, medications for diabetes, htn, and cholesterol . Height and weight were measured using a seca balance scale (seca corp, columbia, md, usa). Body mass index (bmi) was calculated as weight in kg / height in m. bp was measured twice then averaged in participants in a sitting position after a 15-minute rest using a random zero sphygmomanometer (tycos 5090 - 02 welch allyn pocket aneroid sphygmomanometer, arden, nc, usa) and a stethoscope (littmann cardiology, 3 m, st paul, mn, usa). Htn was defined as follows: systolic bp 140 mm hg systolic or diastolic bp 90 mm hg or using antihypertensive treatment.11 dietary intake was measured using a validated semiquantitative food frequency questionnaire (ffq) developed by willett et al.12 this ffq has also been validated by nath and huffman13 exclusively for the cuban american population . Participants self - reported average consumption of specified amounts of various foods over the past year and chose from frequency responses ranging from never to six or more servings per day . In addition to food items, the ffq included questions about type and duration of vitamin / mineral supplement use, alcohol consumption, and specific details about fat, salt, and sugar used in cooking and as condiments . Macro- and micronutrient intake was calculated by multiplying frequency of consumption by the nutrient value of the food item obtained from the harvard university food composition database (boston, ma, usa). Venous blood (20 ml) was collected from each subject after an overnight fast (at least 8 hours) by a certified phlebotomist using standard laboratory techniques . Blood samples were collected into a vacutainer serum separator tube (sst) (becton, dickinson and company, franklin lakes, nj) for analysis of lipids and another tube containing ethylenediamine tetraacetic acid to analyze a1c . After coagulation was completed (3045 minutes), the sst was centrifuged at 2500 rpm for 30 minutes . Lipid panel was assayed by enzymatic methods, and a1c percentages were measured from whole blood samples with the roche tina - quant method by laboratory corporation of america, miami, fl, usa (labcorp). Fresh, single - voided, first morning urine samples were collected from each participant to determine mau by a semi - quantitative assay (immunodip, diagnostic chemicals limited, oxford, ct, usa). Immunodip urinary albumin test uses a monoclonal antibody against human serum albumin to detect mau . The study conducted by davidson et al15 was designed to evaluate the clinical performance of the immunodip dipstick compared with a reference measure recommended by the ada for detecting mau (albumin: creatinine ratio <30 ug / mg [negative];> 30 ug / mg [positive]) determined by laboratory techniques (quest hitachi 717 autoanalyzer). Additionally, results from the immunodip were compared with quantitatively measured albumin concentrations as a secondary outcome . Urinary albumin concentrations were considered <18 mg / l (negative) and> 18 mg / l (positive). Screening for mau with immunodip exhibited a sensitivity of 96% and specificity of 80% when compared with albumin: creatinine ratio> 30 ug / mg . When immunodip was examined against quantitatively measured albumin concentrations, the dipstick yielded a sensitivity of 95% and specificity of 94% . Recommendations from the national academy of clinical biochemistry (nacb) for the diagnosis and management of diabetes indicated that a useful semiquantitative screening test for mau should have a sensitivity> 95%.16 the immunodip dipstick fulfilled the requirements from the nacb as a screening tool to detect mau . In our study, this cut - off value was established by the manufacturer (immunodip, diagnostic chemicals ltd) and corresponded to albumin: creatinine ratio> 30 ug / mg values for mau detection.15 t - tests and chi - square tests were performed to compare means and proportion differences between subjects with and without mau . Unadjusted odds ratios and logistic regression analysis were conducted to investigate the extent to which htn and gc are associated with an increased likelihood of having mau . Controlled variables included in the logistic regression analysis were age, gender, bmi, known duration of diabetes, total cholesterol levels, diabetes and cholesterol medications, smoking, total kcal and protein intake, and intake of potassium, phosphorous, and sodium . Mau was present in 26% (n = 47) of cuban americans with t2d . There was a significantly higher percentage of subjects with mau classified as hypertensive (p = 0.038) and taking diabetes medication (p = 0.039) compared with those without mau . Additionally, subjects who tested positive for mau had significantly higher a1c levels (p = 0.002) than those with normoalbuminuria (table 1). Unadjusted odds ratios indicated that subjects with poor gc were 3.96 times more likely to have positive mau if they had htn compared with those without htn (p = 0.014; 95% confidence interval [ci] 1.25, 12.5) (figure 1). Logistic regression analysis showed that after controlling for covariates, subjects with poor gc were 6.76 times more likely to have mau if they had htn compared with those without htn (p = 0.004; 95% ci 1.83, 23.05) (table 2). The results of this study showed that htn and poor gc are major contributors to increasing the likelihood of having mau among cuban americans with t2d . The combination and/or interaction of these factors over time might increase the risk for progression to proteinuria / esrd and cvd in this population . Our findings are supported by the multicenter study conducted in europe that found that the likelihood of having mau increases in patients when htn and poor gc are present along with other coexisting risk factors for cvd.17 ravid et al5 reported from their longitudinal study that a combination of risk factors, including abnormal bp, plasma cholesterol, a1c levels, high bmi, and male gender, identifies a group of individuals for poor renal and cardiovascular outcomes . Maintaining adequate the uk prospective diabetes study (ukpds),18 a longitudinal study with a 10-year median follow - up, showed that exposure over time to hyperglycemia was associated with diabetes complications in subjects with t2d . This study also indicated that for every 1% reduction in a1c level, the risk for microvascular complications decreased by 37% and diabetes - related death by 21% . A study by thomaseth et al19 showed that in hypertensive t2d subjects with incipient diabetic nephropathy, both tight bp control and optimal gc delay the progression of glomerular filtration rate deterioration . Additionally, hypertensive individuals with t2d under tight bp control experience a reduction in risk for microvascular and macrovascular complications.20 the mechanism and pathways involving mau with diabetic nephropathy and cvd are not fully understood . They may be interrelated with endothelial dysfunction and inflammation.21 stehouwer et al22 examined the relationship between endothelial dysfunction and inflammation with mau and risk of death in a prospective study among subjects with t2d . The results of that study showed that those participants with mau, endothelial dysfunction, and inflammation had an increased risk in mortality; however, the associations of these variables with risk of mortality were independent from each other . Hyperglycemia and obesity were associated with an increase in markers of endothelial dysfunction and inflammation activity,22 possibly indicating that the combinations of these factors may be interrelated in increasing the risk of death . The usual course of mau is progressive; however, not all t2d individuals with mau will develop macroalbuminuria . Several authors have documented remission and/or regression of mau in subjects with t2d.2327 antihypertensive therapy has been shown to reduce or slow the progression of diabetic nephropathy . In a 6-year prospective study, remission and regression to normoalbuminuria were observed in about 50% of t2d individuals.23 other factors independently linked with remission and/or regression of mau were proper bp and gc and short duration of mau . The study by chan et al24 with a 5-year mean follow - up of t2d patients with mau treated with angiotensin - converting enzyme (ace) inhibitor medications showed a 13% reduction in urinary albumin excretion (uae). In addition, the authors pointed out the main role that bp and gc play in renal function . T2d individuals treated with angiotensin receptor blockers (arbs) (eg, irbesartan and valsatran) experienced a 38% and 44% reduction in uae over 2-year and 6-month follow - ups, respectively.25,26 evidence from another prospective study with a 7.8-year follow - up indicated that 30% of the participants on antihypertensive therapy achieved remission to normoalbuminuria.27 additionally, the odds for remission to normoalbuminuria increased in these participants with every 1% decrease in a1c level . Furthermore, in a study conducted by mogensen et al,28 urinary albumin: creatinine ratio was decreased by 50% after a 3-month combination treatment with arb and ace inhibitors in hypertensive t2d individuals with mau . Remission and/or regression of mau may not only conserve renal function but also reduce the risk of cvd . Testing for mau has been recommended by the ada in individuals with t2d to be performed at diagnosis of diabetes and every year afterwards.29 the immunodip dipstick is a rapid and easy screening test to perform, does not involve equipment and/or skilled personnel, exhibits a high sensitivity, requires only a random urine sample (first morning urine is recommended), and has a relatively low cost . However, this test does not quantify urinary albumin values and requires confirmation with a secondary analysis . This method can be used in a physician s office and/or research setting as a first screening tool to detect the presence of mau . The clinical significance of early screening and monitoring for mau is possibly to improve individuals prognosis for microvascular and macrovascular complications, especially among t2d individuals with other concomitant conditions . First, due to the cross - sectional design of the study, single - voided urine was collected to measure mau, and we were not able to determine the cause second, due to the relatively small sample size in this study, our sample of cuban americans with t2d is not representative of the entire cuban american population living in the usa . Nevertheless, to our knowledge, this is the first and only study that has examined the relationship between mau, htn, and gc in this hispanic subgroup, which has an increased risk for t2d and cvd . First, due to the cross - sectional design of the study, single - voided urine was collected to measure mau, and we were not able to determine the cause second, due to the relatively small sample size in this study, our sample of cuban americans with t2d is not representative of the entire cuban american population living in the usa . Nevertheless, to our knowledge, this is the first and only study that has examined the relationship between mau, htn, and gc in this hispanic subgroup, which has an increased risk for t2d and cvd . Early detection of mau in this population may provide more valuable treatment and improve individuals renal and cvd outcomes . The therapeutic goals and strategies for mau should focus on preventing long - term complications associated with t2d, such as kidney and heart diseases . Further investigations need to be carried out to fully understand the mechanism and absolute cvd risk that an individual with t2d has when mau coexists with other comorbidities.

Cancer is the second leading cause of mortality and morbidity in both developed and developing countries . In india, cancer prevalence is estimated around 2.5 million, with over 0.8 million new cases and 0.5 million deaths occurring each year . There is an increase in the incidence of breast cancer and found to be gradually overtaking cancer of the cervix . Breast self - examination (bse) is an important screening measure for detecting breast cancer . There is evidence that women who correctly practice bse monthly are more likely to detect a lump in the early stage of its development, and early diagnosis has been reported to influence early treatment, to yield a better survival rate . Thus the present study aimed at identifying the level of knowledge and practice of bse among degree female students who are the citizen of the future and they can teach their family members, neighbors, friends and the community which helps the people to detect breast cancer in early stage . Thus the morbidity or mortality can be reduced . In the current study only one participant was practicing bse occasionally so incorporating the bse concept in the degree education curriculum is very useful and helpful . The present study aimed at assessing the level of knowledge and the effectiveness of planned teaching program among degree female students on bse . It is a patient - centred, inexpensive and noninvasive method of screening for breast cancer . Based on increased incidence of breast cancer and unawareness of bse among young women, researcher felt a need to provide awareness of bse among young women and can be reduced the incidence and prevalence of breast cancer in future . The objectives of the study were to: to assess the level of knowledge of degree college female students on bse.to determine the effectiveness of planned teaching program among degree college female students on bse.to find the association between pretest knowledge and selected demographical variables . To assess the level of knowledge of degree college female students on bse . To determine the effectiveness of planned teaching program among degree college female students on bse . The study attempted to test following hypotheses all the hypotheses were tested at 0.05 level of significance . There will be a significant difference between pretest and post - test score on knowledge of bse among degree college female students.there will be a significant association between pretest knowledge score and selected demographical variables . There will be a significant difference between pretest and post - test score on knowledge of bse among degree college female students . The study assumed that: the degree college female students will have some knowledge on bse.feel free to express their attitude toward bse.bse helps in early detection of breast cancer . The degree college female students will have some knowledge on bse . Feel free to express their attitude toward bse . Independent variables: teaching program on bse . Selected variables: age, education, parent's education and exposure to mass media . The present study aimed at assessing the level of knowledge and the effectiveness of planned teaching program among degree female students on bse . It is a patient - centred, inexpensive and noninvasive method of screening for breast cancer . Based on increased incidence of breast cancer and unawareness of bse among young women, researcher felt a need to provide awareness of bse among young women and can be reduced the incidence and prevalence of breast cancer in future . The objectives of the study were to: to assess the level of knowledge of degree college female students on bse.to determine the effectiveness of planned teaching program among degree college female students on bse.to find the association between pretest knowledge and selected demographical variables . To assess the level of knowledge of degree college female students on bse . To determine the effectiveness of planned teaching program among degree college female students on bse . The study attempted to test following hypotheses all the hypotheses were tested at 0.05 level of significance . There will be a significant difference between pretest and post - test score on knowledge of bse among degree college female students.there will be a significant association between pretest knowledge score and selected demographical variables . There will be a significant difference between pretest and post - test score on knowledge of bse among degree college female students . The study assumed that: the degree college female students will have some knowledge on bse.feel free to express their attitude toward bse.bse helps in early detection of breast cancer . The degree college female students will have some knowledge on bse . Feel free to express their attitude toward bse . Independent variables: teaching program on bse . Selected variables: age, education, parent's education and exposure to mass media . A pre - experimental one group pretestpost - test study was conducted among degree female students from selected colleges of udupi district . A cluster sampling technique was used to select the college and 40 students selected by convenient method from all the streams of study . Demographic proforma consisted of age, stream of study, education and occupation of parents, income of parents and sources of information on bse . A structured questionnaire consisted of 25 multiple choice questions (mcq) was developed to assess the knowledge on bse . Knowledge scores were categorized into poor (0 - 8), average (9 - 16) and good (17 - 25). Planned teaching program . Lesson plan with power point presentation given for validation contained basic anatomy and physiology, risk factors for breast cancer and steps of bse . Validity of the tools was established by submitting to seven experts and there was 100% agreement on all items . Reliability coefficient of knowledge questionnaire was established by split half method using spearman brown prophecy formula . Data was collected by administering knowledge questionnaire on bse and planned teaching program was introduced for them . On 8 day post - test was done by administering the same tool . The data was analyzed using descriptive (frequency and percentage) and inferential statistics based on the objectives and hypotheses . Demographic proforma consisted of age, stream of study, education and occupation of parents, income of parents and sources of information on bse . A structured questionnaire consisted of 25 multiple choice questions (mcq) was developed to assess the knowledge on bse . Knowledge scores were categorized into poor (0 - 8), average (9 - 16) and good (17 - 25). Planned teaching program . Lesson plan with power point presentation given for validation contained basic anatomy and physiology, risk factors for breast cancer and steps of bse . Validity of the tools was established by submitting to seven experts and there was 100% agreement on all items . Reliability coefficient of knowledge questionnaire was established by split half method using spearman brown prophecy formula . Demographic proforma consisted of age, stream of study, education and occupation of parents, income of parents and sources of information on bse . A structured questionnaire consisted of 25 multiple choice questions (mcq) was developed to assess the knowledge on bse . Knowledge scores were categorized into poor (0 - 8), average (9 - 16) and good (17 - 25). Planned teaching program . Lesson plan with power point presentation given for validation contained basic anatomy and physiology, risk factors for breast cancer and steps of bse . Validity of the tools was established by submitting to seven experts and there was 100% agreement on all items . Reliability coefficient of knowledge questionnaire was established by split half method using spearman brown prophecy formula . Administrative permission was obtained from the principal of selected colleges . Written consent was obtained from the study participants . Data was collected by administering knowledge questionnaire on bse and planned teaching program was introduced for them . On 8 day post - test was done by administering the same tool . The data was analyzed using descriptive (frequency and percentage) and inferential statistics based on the objectives and hypotheses . The data presented in table 1 shows that among 40 samples, the majority (52.5%) of the samples were in the age group of 18 - 19 years and majority (90%) of them were studying in basic science group . Sample characteristics only 35% of them were heard about bse through mass media and 8.5% of participant (only one participant) were practiced bse only one time at the time of study . The description of knowledge scores shows that 72.5% of students had average knowledge on bse in pre test and 85% of students had good knowledge score in post - test [figure 1, table 2]. T test computed to test the effectiveness of planned teaching program on bse (t=12.46) shown in table 3 . Percentage distribution of sample based on knowledge score frequency and percentage of knowledge score computation of effectiveness of planned teaching program the computed to find the association between knowledge and selected variables shows no significant association between knowledge and selected variables . Hence the null hypotheses was accepted and research hypotheses was rejected shown in table 4 . A survey conducted among nurses and midwives in turkey found that among 80 samples only 52% of samples performed bse, no significant relation was found between sociodemographic factor and bse . A study conducted in chennai by s. aruna supported this study as no significant association found between demographic variables and level of knowledge of breast cancer and bse among working women . Study conducted among resettlement colony women, shows that they have poor knowledge on breast cancer and risk factors, warning signs and early detection procedure . The world health organization stresses on promoting awareness in the community and encouraging early diagnosis of breast cancer, especially for women aged 40 - 69 years who are attending primary health care centres of hospitals for other reason, by offering clinical breast examination . There are other many methods to detect the breast cancer but those are expensive for the community . Breast - related matters are sensitive issues for few females; unless the breast lesions starts bothering them they may not seek medical attention . Bse is the cheapest and convenient method to detect the breast cancer in the early stage . In this study majority of the samples acquired good knowledge on bse . Bse plays a major role in early detection and prevention or prompt treatment of breast cancer . By giving teaching to the young girls, they can teach their mother and siblings so that the incidence of the breast cancer may be reduced . It is essential task of each and every woman to do bse and protect herself and her family . The nurse has to play an important role in health promotion and it is only possible when she teaches or educates her client . However, the teaching of bse can help women to be alert to any abnormal changes in their breasts and seek medical advice immediately.

Der p 1 was isolated from house dust mite fecal pellets (allergon) by a multistep procedure 6 involving immunoaffinity chromatography on immobilized anti der p 1 mab (clone 4c1; indoor biotechnologies), removal of contaminating serine proteases on immobilized soybean trypsin inhibitor (sigma chemical co.), and finally fast protein liquid chromatography (fplc) to remove low - molecular - mass contaminants . The purity of the preparation was confirmed by nh2-terminal sequencing on an automatic amino acid sequencer (applied biosystems, inc . ), sds - page analysis (15% gel), and demonstration that enzymatic activity was completely dependent on preactivation with cysteine and totally inhibited by e-64 (l - trans - epoxysuccinyl - leucylamido [4-guanidino]butane). Protein concentration was determined using a bicinchoninic acid (bca) microtiter plate assay and confirmed spectrophotometrically using the empirical absorption coefficient value for der p 1 of e (280 nm) = 16.4 . Before use, der p 1 was preactivated with 5 mm cysteine (sigma chemical co.) to regenerate its thiol group, which becomes oxidized during purification . The catalytic activity of der p 1 was ascertained in a continuous rate (kinetic) assay using the fluorogenic peptide substrate n - tert - butoxy - carbonyl (boc)-gln - ala - arg7-amino-4-methyl - coumarin (amc; reference 6). To block the proteolytic activity of cysteine - activated der p 1, 1,000-fold molar excess of e-64 (sigma chemical co.) was used; a similar molar ratio of the cysteine protease inhibitor iodoacetamide (sigma chemical co.) was used as another sulfhydryl reactive agent . The cells (2 10) were suspended in rpmi (gibco life technologies) and stimulated for 3 d at 37c with con a (5 g / ml final concentration) in the presence of il-2 (100 u / ml) in a humidified atmosphere of 5% co2 . Cd25 cleavage was performed by incubating 10 cells with up to 10 g / ml der p 1 (preactivated with 5 mm cysteine) for 1 h at 37c in a total volume of 200 l serum - free aim v medium (gibco life technologies). The cells were then resuspended in rpmi containing 2% fcs, stained for 45 min at room temperature in the dark with fitc - labeled anti mouse cd25 mab (clone amt-13; sigma chemical co.), and fixed with 5% formaldehyde . The expression of other t cell surface markers, namely cd3, cd4, and cd8, was monitored in the same way using appropriate pe- or fitc - labeled antibodies (clone kt3, beckman coulter; and clones yts191.1 and kt15 [serotec ltd . Five groups of 10 female cba / j mice were given six weekly intraperitoneal injections of 10 g of proteolytically active der p 1, 10 g of e-64blocked der p 1, 10 g of iodoacetamide - blocked der p 1, 10 g of ova (as proteolytically inactive antigen; sigma chemical co.), or 10 g of ova with e-64, respectively . A tail bleed was obtained 1 wk before the start of immunization (prebleed), and a total bleed was obtained by cardiac puncture 1 wk after the last injection (final bleed). The proteolytic activity of der p 1 and its inhibition with e-64 or iodoacetamide in the immunization mixture were ascertained as described above . Serum samples were initially titrated to determine the optimal dilution for testing each antibody isotype and subclass . The optimal dilutions used here were 1/10 for detecting total ige, der p 1specific ige, and ova - specific ige and 1/20,000, 1/40,000, and 1/250 for detecting der p 1specific igg, igg1, and igg2b, respectively . Mouse ige (clone r35 - 72; pharmingen) as capture antibody and a second biotinylated monoclonal anti der p 1specific ige, ova - specific ige (measured using samples that have been depleted of igg on a protein g column [pharmacia]), and der p 1specific igg, igg1, and igg2b were detected on microtiter plates coated with a 4 g / ml solution of either der p 1 or ova and developed with biotinylated (for ige clone r35 - 118 and igg1 clone a85 - 1 [pharmingen] and for igg2b clone ab275 [the binding site]) or alkaline phosphatase conjugated isotype - specific antibodies (for igg; sigma chemical co.). Alkaline phosphatase conjugated extravidine (sigma chemical co.) was used in conjunction with biotinylated antibodies . Unpaired student's t test was used to compare levels of antibody responses between the different immunization groups; p <0.05 was considered significant . Der p 1 was isolated from house dust mite fecal pellets (allergon) by a multistep procedure 6 involving immunoaffinity chromatography on immobilized anti der p 1 mab (clone 4c1; indoor biotechnologies), removal of contaminating serine proteases on immobilized soybean trypsin inhibitor (sigma chemical co.), and finally fast protein liquid chromatography (fplc) to remove low - molecular - mass contaminants . The purity of the preparation was confirmed by nh2-terminal sequencing on an automatic amino acid sequencer (applied biosystems, inc . ), sds - page analysis (15% gel), and demonstration that enzymatic activity was completely dependent on preactivation with cysteine and totally inhibited by e-64 (l - trans - epoxysuccinyl - leucylamido [4-guanidino]butane). Protein concentration was determined using a bicinchoninic acid (bca) microtiter plate assay and confirmed spectrophotometrically using the empirical absorption coefficient value for der p 1 of e (280 nm) = 16.4 . Before use, der p 1 was preactivated with 5 mm cysteine (sigma chemical co.) to regenerate its thiol group, which becomes oxidized during purification . The catalytic activity of der p 1 was ascertained in a continuous rate (kinetic) assay using the fluorogenic peptide substrate n - tert - butoxy - carbonyl (boc)-gln - ala - arg7-amino-4-methyl - coumarin (amc; reference 6). To block the proteolytic activity of cysteine - activated der p 1, 1,000-fold molar excess of e-64 (sigma chemical co.) was used; a similar molar ratio of the cysteine protease inhibitor iodoacetamide (sigma chemical co.) was used as another sulfhydryl reactive agent . Spleen t cells were obtained from c57bl/6j mice using standard procedures . The cells (2 10) were suspended in rpmi (gibco life technologies) and stimulated for 3 d at 37c with con a (5 g / ml final concentration) in the presence of il-2 (100 u / ml) in a humidified atmosphere of 5% co2 . Cd25 cleavage was performed by incubating 10 cells with up to 10 g / ml der p 1 (preactivated with 5 mm cysteine) for 1 h at 37c in a total volume of 200 l serum - free aim v medium (gibco life technologies). The cells were then resuspended in rpmi containing 2% fcs, stained for 45 min at room temperature in the dark with fitc - labeled anti mouse cd25 mab (clone amt-13; sigma chemical co.), and fixed with 5% formaldehyde . The expression of other t cell surface markers, namely cd3, cd4, and cd8, was monitored in the same way using appropriate pe- or fitc - labeled antibodies (clone kt3, beckman coulter; and clones yts191.1 and kt15 [serotec ltd . Five groups of 10 female cba / j mice were given six weekly intraperitoneal injections of 10 g of proteolytically active der p 1, 10 g of e-64blocked der p 1, 10 g of iodoacetamide - blocked der p 1, 10 g of ova (as proteolytically inactive antigen; sigma chemical co.), or 10 g of ova with e-64, respectively . A tail bleed was obtained 1 wk before the start of immunization (prebleed), and a total bleed was obtained by cardiac puncture 1 wk after the last injection (final bleed). The proteolytic activity of der p 1 and its inhibition with e-64 or iodoacetamide in the immunization mixture were ascertained as described above . Serum samples were initially titrated to determine the optimal dilution for testing each antibody isotype and subclass . The optimal dilutions used here were 1/10 for detecting total ige, der p 1specific ige, and ova - specific ige and 1/20,000, 1/40,000, and 1/250 for detecting der p 1specific igg, igg1, and igg2b, respectively . Mouse ige (clone r35 - 72; pharmingen) as capture antibody and a second biotinylated monoclonal anti der p 1specific ige, ova - specific ige (measured using samples that have been depleted of igg on a protein g column [pharmacia]), and der p 1specific igg, igg1, and igg2b were detected on microtiter plates coated with a 4 g / ml solution of either der p 1 or ova and developed with biotinylated (for ige clone r35 - 118 and igg1 clone a85 - 1 [pharmingen] and for igg2b clone ab275 [the binding site]) or alkaline phosphatase conjugated isotype - specific antibodies (for igg; sigma chemical co.). Alkaline phosphatase conjugated extravidine (sigma chemical co.) was used in conjunction with biotinylated antibodies . Unpaired student's t test was used to compare levels of antibody responses between the different immunization groups; p <0.05 was considered significant . Der p 1 is a 25-kd cysteine protease whose structure has been modeled 7 on the crystal structure of papain, with which it shows considerable sequence similarities, most notably for residues involved in the enzyme active site 8 . The proteolytic activity of der p 1 can be inhibited by e-64, the class - specific inhibitor of microbial origin 9 . This inhibition is brought about when cysteine within the der p 1 active site forms a thioether covalent bond with the epoxy group of e-64 . This is an irreversible process that does not lead to significant structural changes, as evidenced by crystallographic studies of a papain e-64 complex 10 . We have purified der p 1 from fecal pellets using a multistep procedure and confirmed its purity by nh2-terminal sequencing, sds - page analysis, and demonstration that enzymatic activity was completely dependent on preactivation with cysteine and inhibited by e-64 and iodoacetamide (fig . 1). We have recently shown that der p 1 selectively cleaves human cd25 from the surfaces of peripheral blood t cells 4 . Here we demonstrate that der p 1 also selectively cleaves cd25 from cultured mouse spleen t cells (fig . 2), which is not surprising given the high degree of sequence homology that exists between human 11 and mouse 12 cd25 . This observation has therefore provided the justification for using this animal species for testing our hypothesis, namely that the proteolytic activity of der p 1 is a major contributor to its allergenicity . Intraperitoneal immunization of groups of 10 cba / j mice with either proteolytically active or inactive (e-64blocked) der p 1 over a 6-wk period showed a statistically significant enhancement in total ige (p <0.01) and der p 1specific ige (p <0.02) responses in animals immunized with proteolytically active der p 1 . This effect was ige specific, as der p 1specific igg, igg1, and igg2b responses increased to the same extent with proteolytically active or inactive der p 1 (fig . We are not sure why the igg1 response did not follow that of ige, as these two isotypes are considered to be coregulated in the mouse . However, the ige - restricted enhancement seen in response to immunization with proteolytically active der p 1 does suggest a mechanism that is unique to ige isotype switching / synthesis . Furthermore, our control experiments clearly show that the ige - specific effect observed here is not due to e-64 exerting a suppressive influence on ige production by a mechanism that is independent of its binding to the der p 1 enzyme active site (fig ., suppression of total ige (p <0.04) and der p 1specific ige (p <0.05) productions was also obtained when the proteolytic activity of der p 1 was blocked with iodoacetamide, another sulfhydryl reactive agent . Second, the ige antibody response to ova, a proteolytically inactive antigen, was not suppressed when the animals were immunized with ova plus e-64 . Our results are direct evidence that the cysteine protease activity of der p 1 induces a significant increase in ige responses . Such an effect is clearly consistent with the ability of der p 1 to proteolytically cleave mouse cd25 and induce a th2 response by modulating the balance between il-4 and ifn- 13 . The recent demonstration in mice that leishmania mexicana cysteine proteinase deficient mutants potentiate a th1 response, compared with the th2 response normally seen in response to infection with wild - type parasite 14, is also of great relevance here . Allergic individuals could potentially be achieved by administering der p 1 in a catalytically inactive (mutant) form . On the other hand, exploring the potential th2 adjuvant property of the proteolytic activity of der p 1 would have important implications in defining principles for modulation of th1-mediated pathological conditions . Our demonstration that the cysteine protease activity of der p 1 enhances total ige production, apart from increasing der p 1-specific ige, suggests that this allergen may play a central role in destabilizing the microenvironment within target tissues to one that is proallergic and thus aids in the initiation and propagation of the allergic cascade . In other words, the proteolytic activity of der p 1 may exert an ige - specific adjuvant effect . The in vivo relevance of the proteolytic activity of der p 1 is further highlighted by reports demonstrating that it increases the permeability of the human respiratory epithelium to macromolecules 1516 . Such observations, together with our current findings showing a direct effect on the immune system, indicate that the proteolytic activity of der p 1 is a major contributor to its allergenicity.

Given the risk of paralysis associated with cervical transforaminal injection, is it time to reconsider transforaminal injections of the lumbar spine? Arguments for discontinuing lumbar injections have been discussed in the anesthesia literature, raising concern about the risks of epidural steroid injections (esis). In a 47-year - old man, paraplegia of the lower extremities developed, specifically conus medullaris syndrome, after he underwent an esi for recurrent pain . The patient felt his legs going dead; paraplegia of the lower extremities was noted . An initial magnetic resonance imaging study performed after the patient was transferred to the emergency department was unremarkable . However, a later neurosurgical evaluation showed conus medullaris syndrome, and a second magnetic resonance imaging study showed the conus infarct . We conducted a search of the pubmed database of articles from 2002 to 2011 containing the following keywords: complications, lumbar epidural steroid injection(s), cauda equina syndrome, conus medullaris infarction, spinal cord infarction, spinal cord injury, paralysis, paresis, plegia, paresthesia, and anesthesia . Summarizing this case and 5 similar cases, we weigh the potential benefits and risks of esi . Although one can safely assume that this severe, devastating complication is rare, we speculate that its true incidence remains unknown, possibly because of medicolegal implications . We believe that the rarity of this complication should not preclude the continued use of transforaminal esi; rather, it should be emphasized for discussion with patients during the consent process . A 47-year - old man who had undergone an l4/l5 laminectomy and discectomy 5 years earlier presented with recurrent pain in the left buttock and posterior leg . After receiving an esi by a local anesthesiology group within the cincinnati, ohio, area, the patient was transferred from the outpatient pain clinic to our institution . Information regarding his medical history and prior treatment, as well as details of the procedure, was limited to that provided by the patient himself or the accompanying procedure report . The patient reported that he had previously received esis on the left side of his lower back; this was the second injection in a series of 3 . Correct needle placement in the left l5-s1 neural foramen was verified with epidurography immediately after the injection, the patient felt his legs going dead; paraplegia of the lower extremities was noted . Because of the concern for intrathecal injection with resultant motor blockade, the patient was monitored for 4 hours . When no clinical improvement was observed, he was transferred to a nearby community hospital emergency department for neurologic examination . Although the initial magnetic resonance imaging (mri) study performed in the emergency department was unremarkable (fig . However, a second mri study to evaluate the possibility of vascular complications obtained 48 hours after the injection showed a conus infarct (fig . 2). Five hours after esi in a 47-year - old man, magnetic resonance imaging scans performed at the emergency department were unremarkable, showing a normal appearing conus . (a) (b) t1-weighted sagittal image (repetition time, 416.7; echo time, 15.0). Magnetic resonance images (about 48 hours after the procedure) showing extensive signal abnormalities within the lower thoracic spinal cord and conus compatible with the clinical diagnosis of conus infarct . (a) t1-weighted sagittal image (repetition time, 675.0; echo time, 9.6). (b) t2-weighted sagittal image (repetition time, 3640.0; echo time, 102.0). (c) short tau inversion recovery (stir) sagittal image (repetition time, 4000.0; echo time, 58.0). At 1-month follow - up, the patient could walk without assistance with a slow and calculated gait, had symmetric lower - extremity strength, and had dorsiflexion strength of 4 of 5 bilaterally . His urinary urge sensation had returned, and occasional episodes of fecal incontinence occurred in relation to bladder overdistention . Our search of the pubmed database (20022011) included the keywords complications, lumbar epidural steroid injection(s), cauda equina syndrome, conus medullaris infarction, spinal cord infarction, spinal cord injury, paralysis, paresis, plegia, paresthesia, and anesthesia . Esi is regarded as an effective and conservative means of treating low - back pain resulting from nerve root inflammation . Recent case reports of post - procedural conus medullaris syndrome after spinal vascular compromise raise the question regarding safety . Our case represents a sixth patient in whom conus medullaris syndrome developed after sustaining an acute spinal cord infarct during esi . In 5 other case reports (table 1), this complication affected patients (aged 4271 years) who had previous lumbar surgery and then underwent a transforaminal steroid injection . Of note, findings of spinal cord injury were not detectable on the initial mri study within the first 24 hours in 1 case . Injections are typically evaluated first by aspiration and injection of contrast . A nonvascular penetrating injection of contrast media verifies safe needle placement . However, in a series of 761 lumbosacral transforaminal injections performed, furman et al . Reported that the sensitivity of positive flash or actual aspiration was only 44.7%, because only 38 of 85 patients had shown either flash or actual aspiration before injection . Specifically, they showed that there was an 11.2% rate of vascular injection in their series . In evaluating correct placement of 316 caudal - approach esis, implicated aspiration as an errorprone method for verifying needle placement, reporting a 9.2% incidence of vascular violation despite negative findings on aspiration . Blood is supplied to the caudal - most portion of the spinal cord by the anterior spinal artery, 2 posterior spinal arteries, segmental radiculomedullary arterial branches, and most importantly, the artery of adamkiewicz . The location of the artery of adamkiewicz, which is the primary blood supply to the conus medullaris, is fairly unpredictable . It travels with the nerve through the foramen at or near the level of its origin from the thoracolumbar segmental arteries . . Showed that the artery of adamkiewicz originated from the left t9 - 12 posterior intercostal arteries in 75% of cases and from the l1 - 2 lumbar arteries in 10% of cases . In 31 cadavers biglioli et al . Located this artery between t12 and l3 in 26 cases (83.9%). In a 2002 review of more than 4000 spinal angiograms, lo et al . Examined the variability of the artery of adamkiewicz, noting that it originated at l2 in 1% of cases and l4 in 0.075% of cases . Of the proposed mechanisms by which spinal cord medullary infarction may occur, one explanation may be the combined effect of an undetected direct arterial injection into a low - lying artery of adamkiewicz and the resultant embolic incident from the injectate . Houten and errico proposed that the collaterals surrounding the cord at the level of this artery were proximal to the injection site and thus allowed direct passage of the injected material into the conus . However, as discussed by lo et al . Considering this observation, we believe that it is unlikely that our patient had this low - lying artery that could have then been injected with an epidural steroid at this precise location . Another plausible explanation is that an inadvertent sacral radicular artery injection carried injection material distally to the spinal cord . Reported that material injected into the abdominal aorta below the level of the artery of adamkiewicz would appear in the conus through collateralization . The most likely cause is injection of steroid particulate through either the artery of adamkiewicz or collateral radiculomedullary arterial branches . Prior lumbar surgery may lead to compromise of normal vascular supply to the spinal cord, thus making it more susceptible to vascular injury . Subsequent thrombus formation would result in an embolic infarct in the spinal cord supplied by the affected artery . Florey noted that such vasoconstriction was a local effect lasting 5 seconds to 10 minutes at the site of injury only . Found that fine needle (30-gauge) arterial puncture in rhesus monkeys induced intense vasospasm, typically lasting anywhere from 4 hours to 4 days . Many studies have examined mechanically induced vasospasm of the cerebral vasculature and may be indirectly suggestive that a similar phenomenon can occur in the spinal cord and conus medullaris . Of the multiple recommendations made to avoid such devastating complications as paralysis, first and foremost is the strict adherence to the standard and widely accepted techniques of transforaminal esis . These guidelines include the use of multiplanar fluoroscopy or computed tomography guidance together with contrast material to prevent complications . If recovery of neurologic function fails to occur in a patient within a 2- to 3-hour time period after esi, an initial mri study may be obtained to exclude an epidural hematoma . Repeat mri after 24 hours should be performed as well . In our patient, as well as 1 other reported case, a delayed effect was observed with mri signal changes occurring after 24 hours . Given the risk of paralysis associated with cervical transforaminal injection, is it time to reconsider transforaminal injections of the lumbar spine? Although the risk of permanent neurologic deficit is negligible, arguments for discontinuing lumbar injections have been presented in the anesthesia literature . However, when one considers the potential benefit of this noninvasive treatment modality, abandoning such injections may be premature . However, we speculate that more than 6 cases of conus medullaris syndrome after esi have occurred, and medicolegal considerations may explain why they may go unreported . At the very least, patients should be clearly informed regarding the potential risks of esi, including paralysis . Although one can safely assume that this severe, devastating complication is rare, its true incidence remains unknown . In our opinion, the rarity of this complication should, at this time, not preclude the continued use of transforaminal esi for relief of pain in select patients but should be included in the consent process.

Alzheimer's disease (ad) is a neurodegenerative disorder that is clinically characterized by progressive mental decline and histopathologically defined by highly abundant amyloid deposits and neurofibrillary tangles in the brain parenchyma . The identification of mutations within the amyloid precursor protein (app) and presenilin (ps) genes that cause autosomal dominantly inherited ad and that result in increased production of amyloid - prone forms of a established beyond doubt that the processing of app and the production of a peptides are intimately involved in the disease process and led to the proposal and the reinforcement of the alzheimer amyloid cascade hypothesis [1, 2]. The role of amyloid in neuronal dysfunction has recently been extended by the discovery of small, soluble, oligomers of the a peptide, some forms of which have been termed addls (a-derived diffusible ligands), protofibrils, or a*56 [36]. These a oligomers are not only potential intermediates in the formation of amyloid filaments, but they also have been shown to be neurotoxic themselves and to inhibit long - term potentiation (ltp), a cellular model of memory, in hippocampal slices [4, 7, 8]. Thus, the amyloid cascade hypothesis now includes the essential role of a oligomers in the neurodegeneration process . Despite its strength, the amyloid cascade hypothesis is incomplete without including the essential role of amyloid - associated inflammatory proteins . For example, biochemical and histological studies first showed that, in addition to a, amyloid deposits also contained the inflammation / acute phase protein 1-antichymotrypsin (act) and, later, apolipoprotein e (apoe) [10, 11], which were both hypothesized to serve as catalysts or pathological chaperones of amyloid formation [9, 11, 12]. These and other results also indicated that alzheimer's disease and its manifestation in middle - aged down syndrome may include an inflammatory process, for both act and apoe are inflammatory and/or acute phase proteins in other contexts, and both are overexpressed in affected regions of the ad brain (for reviews see [1315]). Indeed, alzheimer himself first identified the inflammatory component of alzheimer's disease when he described reactive astrocytes and microglia in affected brain regions of his first patient . However, until inflammatory proteins such as act, il-1, hla, and apoe were found to be overexpressed in ad and ds brains, the term inflammation was explicitly excluded from the clinical and pathological description of ad because of the lack of edema and lymphocyte infiltration [911, 17, 18]. The significance of these biochemical results instigated and was reinforced by parallel genetic discoveries implicating a role for inflammation in ad . In particular, inheritance of the apoe 4 allele was found to be the strongest known risk factor for ad besides age, with one copy increasing ad risk 35-fold and two copies over 10-fold [1921]. Because of apoe's essential genetic, and therefore presumably biochemical, contribution to ad pathology and cognitive decline, it is critical that its role in the ad pathogenic pathway / amyloid cascade be elucidated in order for therapeutics based on apoe to be designed . While recent excellent and encyclopedic literature reviews describe the many potential roles that apoe plays in ad [2326], this focused review will concentrate on the interaction between a and apoe and other inflammatory proteins, on the effects of such interactions, and on their implications for designing apoe - based ad therapies . The central question we try to answer is whether increasing or decreasing apoe level and/or function will serve best to reduce ad / ds pathology and cognitive decline . Lack of a clear answer may lead to the development of drugs that, rather than serving as an ad therapy, instead potentially exacerbate the disease . To determine whether inflammation contributes to alzheimer's disease rather than being merely a correlative pathological feature in the ad brain, we and others tested the hypothesis that act and/or apoe serve as amyloid catalysts or pathological chaperones . Numerous in vitro and in vivo studies showed that mature amyloid deposition and the associated cognitive decline is strongly stimulated by apoe and act in a dose - dependent and isoform - specific manner, with apoe4 being the strongest promoter of a polymerization and apoe2 being an inhibitor, paralleling the effect of these two isoforms in humans [2738]. Indeed, without one or the other of these amyloid catalysts expressed in the brain, amyloid deposition is profoundly delayed in app transgenic mice and does not become filamentous . Such app+/apoe ko animals also exhibit normal cognition despite levels of a expression equal to the apoe - expressing app animals . Elegant work by manelli and colleagues also showed that native lipidated apoe4 from transgene replacement astrocytes increases a neurotoxicity compared to apoe3 or e2, indicating that apoe4 provides a negative gain of function . Finally, jones and colleagues recently showed that apoe4 also promotes the conversion and enhanced synaptic localization of a as oligomers, the most neurotoxic form of the alzheimer amyloid peptide [40, 41]. These recent studies extended prior work showing that apoe copurifies with a during biochemical isolation of amyloid from human brains, and that apoe preferentially interacts with a peptides in a -sheet structure [4245]. Together these results show that inflammatory proteins, particularly apoe, are integral parts of the amyloid cascade, and that without them the cascade would be arrested at the level of the harmless a monomer, and no ad would ensue . The view of apoe as an integral and pathological part of the amyloid cascade has been shaken by experiments that suggest that apoe, far from being an amyloid catalyst, serves to clear a from the brain . Under this view, apoe is protective, with human apoe4 being less protective than apoe3 or e2 (for the most recent discussion, see and commentary at http://www.alzforum.com/). The first experiments that suggested apoe's role as a neuroprotector examined the pathology and cognition of app transgenic mice carrying a second transgene expressing one or another human apoe isoform . Contrary to expectations, amyloid deposition in these mice was inhibited by the human apoe transgene, as though human apoe was protective . Ultimately, the mice did develop amyloid, with the apoe4-expressing strain accumulating earlier and more extensive pathology [33, 34, 48, 49]. It was proposed that human apoe might serve to inhibit a clearance from the brain compared to mouse apoe, with apoe4 inhibiting clearance the most . Other experiments showed that indeed, clearance of a species was inhibited by complexing with apoe, especially apoe4 [46, 50]. The possibility that interaction with apoe modulated an a clearance mechanism appeared to be supported by the finding that introduction of anti - a antibodies or other a-binding proteins such as gelsolin, led to a reduced amyloid load in the brain and rapidly improved cognition, with little evidence of a-binding agents invading the brain parenchyma [4154]. We also introduced apoe itself into the circulation via parabiosis and found that it induced amyloid clearance without entering the brain in ad model mice . Thus the peripheral sink hypothesis became a viable alternative or addition to the amyloid cascade hypothesis, with apoe potentially playing an additional role as an a-binding peripheral protein . Most recently, an approach to therapy has been investigated in ad mice that is based on activating the liver x receptor (lxr), which also exists on other cells including microglia [5557]. Activation of lxr results in increased expression of many proteins including apoe and its lipidating enzyme, atp - binding cassette transporter a1 (abca1). The results indicate that activating lxr with the ligand gw3965 or the fda - approved antiskin cancer drug bexarotene reduces soluble and insoluble a and improves cognition in app tg mice, while knocking out the abca1 gene in app mice showed a tendency to reduced amyloid load . Because apoe expression and lipidation is stimulated by lxr activation, the results were interpreted as proof that increased apoe levels help microglia clear a and amyloid, as indeed some earlier cell culture experiments had suggested . However, it has also been shown that genetic overexpression of abca1 reduces amyloid deposition in mice where the apoe levels are unchanged . Hence, because lxr stimulation influences the levels of many proteins, it is problematic to definitively link its in vivo action to the altered level of one particular protein . Furthermore, the increased levels of abca1 induced by bexarotene enhance apoe lipidation, a change that is known to alter apoe / a interactions . Hence, it is important to consider the lipidation state of apoe, which affects its function, in addition to the absolute levels of apoe . When trying to distinguish and weigh the value of two hypotheses, it is instructive to consider their testable predictions . If apoe is an amyloid catalyst, then reducing apoe levels or function in the brain should result in reduced amyloid deposition and reduced cognitive decline . If on the other hand, apoe is involved in a clearance with human apoe4 being a greater inhibitor of clearance (or poorer clearer), then reducing apoe levels or apoe binding to a should increase amyloid deposition and cognitive decline . All experiments carried out so far in vitro or in transgenic mice indicate that the ability of a to form neurotoxic filaments or oligomers and cause cognitive decline are increased in the presence of apoe, particularly mouse apoe and human apoe4, with apoe2 being protective . In contrast, in the complete absence of apoe, the mutant app gene and its product a are harmless, generating neither amyloid deposits, synaptic disfunction, or cognitive decline, with one copy of apoe having an intermediate effect, as discussed above . The in vitro experiments in particular indicate that apoe likely acts catalytically to promote a polymerization, as the molar ratio of a to apoe of about 200/1 was appropriate for the formation of neurotoxic products [2730]. Most recently, earlier work showing that mice expressing only one apoe gene accumulated less amyloid than those with two apoe genes (32) was repeated in two different laboratories using human apoe knock - in mice, and the same result was found, that is, lower doses of apoe3 or apoe4 led to reduced amyloid deposition [59, 60]. The simplest interpretation of the in vitro, cell culture, and transgenic mouse data is that apoe is necessary for a to polymerize into neurotoxic oliogomers / filaments, probably by binding to a and thus altering its structure more toward the -sheet and more easily allowing successive a peptides to add on to the growing chain . The recent finding that apoe promotes a oligomer formation in vivo reinforces this interpretation [40, 41]. Whether apoe is only needed to initiate the polymerization or also to prepare each peptide for addition to the growing filament is not yet known . Even though the key predictions of the polymerization hypothesis, that is apoe serving as an a filament catalyst, have been borne out, the compelling experiments demonstrating that human apoe inhibits filament formation in a mouse background require explanation . Furthermore, data from ladu and colleagues and by others have shown that lipidated apoe, presumably the prevalent form in vivo, binds a with an affinity of e2> e3 finally, the elegant and thorough experiments of castellano and colleagues show very convincingly that expression of a human apoe4 transgene (in the absence of mouse apoe) leads to a longer half - life, (i.e., slower clearance) of a in the brain interstitial fluid compared to e2 or e3 . The apoe - a binding studies might be interpreted as support for apoe functioning in a clearance because apoe2, for example, would bind a tightly and could thereby promote its removal from the interstitial fluid via lrp receptors [50, 6164]. However, an important feature of any catalyst is that it must bind its substrate only tightly enough to convert it to the transition state structure and then release it as the reaction is completed [65, 66]. If a mutation leads to an overly tight substrate binding, then no further reaction can occur . Thus apoe2 could indeed bind a most tightly, and thereby not only prevent apoe4 from binding and promoting a oligo / polymerization, but also prevent the spontaneous polymerization of the peptide . The ability of different apoe isoforms to bind a with different strengths can also explain why human apoe isoforms slow amyloid deposition in the presence of the endogenous mouse apoe, for they may bind a more tightly or differently than mouse apoe and slow the catalytic conversion of a into oligomers / polymers in the mouse background . The data showing that human apoe inhibits a clearance can also be interpreted as reflecting apoe's role in catalyzing a oligo / polymerization . Pathologic macromolecular structures are often resistant to various clearance mechanisms designed for monomeric species, whether by intracellular proteasome degradation or cross - membrane / bbb transfer, thus allowing their accumulation . Only when oligo / polymeric structures are anticipated and physiological clearance mechanisms are in place to handle them, as for antibody - antigen complexes, will clearance be facilitated by conversion to larger structures . Because apoe clearly has the ability to catalyze the conversion of a into oligomeric and polymeric structures, it is reasonable to assume that those structures will be more difficult to clear, and that such difficulty will be detected as clearance inhibition in the brain, for instance, by apoe4, in pulse chase type experiments, while the higher apoe levels in blood may aid the clearance of a from the circulations (figure 1). Finally, the ability of gw3965 and bexarotene to reduce soluble and insoluble a in the brain of tg app mice and improve cognition is most easily understood as resulting from a general activation of the phagocytic activity of microglia . Previous work showed that activation of microglia by acute intracerebral treatment of app mice with lps or with granulocyte - macrophage stimulating factor can similarly reduce amyloid load and improve cognition [6769] but that long - term peripheral treatment with lps exacerbated amyloid deposition in an apoe - dependent manner . Stimulation of microglial activity via induction of toll - like receptor 9 (tlr9) has also been shown to greatly reduce amyloid load and improve cognition . Clearly the interaction of neuroinflammation, microglia, and amyloid load is complex, and the fact that bexarotene cures ad in mice is more likely to be despite, rather than because it stimulates expression of apoe . A good test of any hypothesis about the pathogenesis of a disease is whether it successfully predicts how the pathogenesis can be inhibited or reversed . For example, small fragments of a corresponding to the amino acid sequences to which act (a1 - 12) and apoe (a12 - 28) bind can serve as decoy peptides that prevent the binding of apoe to a and its catalysis of a into neurotoxic species . This early in vitro work has recently been repeated and confirmed in other laboratories [72, 73]. The decoy principal was extended in vivo by preparing a version of a12 - 28 that has a better plasma 1/2 life and is nonfibrillogenic / nontoxic . It was shown that this peptide could be peripherally introduced into a transgenic app mouse, where it effectively entered the brain and prevented / reversed oligomer formation, amyloid deposition, and cognitive decline [7476]. Similarly, amyloid plaques in app mice contain mouse act and injecting a1 - 11 into one side of the app mouse brain to block act's binding site with endogenous a rapidly reduced amyloid load compared to the other, vehicle - injected side of the brain . Furthermore the inflammatory cytokine il-1 that is overexpressed in ad brain induces astrocyte expression of act, and blocking il-1 expression in app transgenic mice by ibuprofen treatment, thereby reducing mouse act expression, lowers amyloid formation and restores cognition . Evidently, blocking act or apoe expression or function, both in vitro or in vivo, successfully prevents a pathology and neurotoxicity . Apolipoprotein j also binds a and can be shown to aid its passage across the blood brain barrier [7983]. Interestingly, knocking out either apoj or apoe reduces amyloid deposition in app transgenic animals, yet knocking out both leads to robust amyloid deposition at an even earlier age than arises in nonmanipulated app animals . This result may reflect the ability of mouse act to promote amyloid formation, but that in the presence of the stronger binding apoe and apoj proteins mouse act is prevented from exhibiting its catalytic activity . The role of apoe and act in the alzheimer pathogenic pathway has potentially general implications . One of the most studied classes of a binding proteins are specific anti - a antibodies, which form the basis of both passive and active immunization therapies for alzheimer's disease (for review see). The finding that apoe and act can catalyze a oligo / polymerization begs the question of whether a antibodies might also promote or inhibit a polymerization . Indeed we found that two a antibodies, 6e10 which is directed to the same the n - terminal sequence bound by act, and 13 m, which binds to the c - terminus, function very differently in the in vitro a polymerization assay . 6e10 inhibits act - catalyzed polymerization of a while 13 m inhibits act catalysis much less and even promotes some polymerization itself . Interestingly, the n - terminus of a is also the target of many attempts at ad immunotherapy with the aim of inducing microglial phagocytosis of neurotoxic a species . Yet removing the microglial - binding fc portion of 3d6 antibodies to a1 - 5 to generate fab'2 fragments does not reduce the antibody's ability to remove diffuse amyloid in app mice . Evidently, only its a-binding feature is required to allow the antibody to remove amyloid . A possible explanation for this result is that the antibody functions by blocking a interaction with mouse act . The consequent suppression of act - catalyzed oligo / polymerization could thus tilt the dynamic process of plaque development toward depolymerization . These results illustrate the fact that a-binding proteins can have multiple effects on polymerization and that their full range of activities must be considered when using them as potential targets or tools for therapeutic intervention . Although a oligomers have been shown to be highly neurotoxic in vitro and in vivo, and their formation is promoted by apoe4, the mechanism of their toxicity is still being elucidated . The data reviewed above coupled to other recent findings suggest a novel mechanism for a toxicity that encompasses the essential role of apoe . Specifically, a oligomers bind to and inhibit certain microtubule motors that are essential for the function and stability of the mitotic spindle similar motors, including kinesin 5, are also present in mature neurons [88, 89]. We have found recently that inhibition of mt motor function by a or by the specific kinesin 5 inhibitor monastrol prevents the efficient transport of receptors such as the ldlr, the nmda neurotransmitter receptor, and the p75 neurotrophin receptor to the cell surface, resulting in reduced function (; in preparation). Similarly, apoe, particularly apoe4, has been shown to reduce the cell surface levels and function of nmda, ampa, and apoer2 receptors in neurons . This latter finding can now be understood as potentially reflecting the ability of apoe4 to promote the conversion of endogenous neuronal a into oligomers, which then inhibit mt - based transport of key cellular components such as receptors to their functional location . In sum, it appears that the preponderance of the data can be most consistently interpreted as showing that the brain inflammatory protein apoe plays a catalytic role in the ad / ds amyloid cascade and consequent cognitive decline, with binding and clearance differences between the apoe isoforms reflecting their differing abilities to bind to a and catalyze its conversion into neurotoxic macromolecular species (figure 2). This conclusion, and the in vivo demonstration that blocking apoe - a interaction prevents ad in a mouse model, suggests that this decoy approach should be translatable into human patients and serve as an effective new approach to ad therapy . Other a-binding proteins may be similarly manipulated by a decoy approach to reduce oligomerization and polymerization of a into neurotoxic species . However, the finding that different antibodies to a can both inhibit act - catalyzed a polymerization and promote polymerization of a itself, argues that immunotherapy must be approached with care to avoid the use or induction of antibodies that can catalyze further oligo / polymerization of a, instead of inducing its phagocytosis and removal . Furthermore, human and mouse intracerebral environments may differ in important ways with respect to the pattern and activities of a-binding proteins and may also respond differently to intervention or inflammation . Such differences may explain why so many treatments that were successful in reducing amyloid - dependent cognitive decline in transgenic mice have failed to translate into human ad patients . Finally, the ability of a oligomers to inhibit key microtubule motors and prevent the transport of neurotrophin, neurotransmitter, and other receptors to the cell surface may underlie their neuronal toxicity . It is apparently the apoe-, especially e4-dependent formation of such a oligomers, that constitutes the key catalytic step in the ad pathogenic pathway.

Musculoskeletal tissues show increased bone fragility, loss of cartilage resilience, reduced ligament elasticity, loss of muscular strength, and fat redistribution decreasing the ability of the tissues to carry out their normal functions . The loss of mobility and physical independence resulting from arthropathies and fractures can be particularly devastating in this population, not just physically and psychologically, but also in terms of increased mortality rates . The aim of this article is to present some of the most frequent musculoskeletal disorders of the elderly, including some misleading presentations . We used the following terms and some of its combinations: elderly, aged, epidemiology, fracture, osteoporosis, vertebral, hip, pelvis, arthritis, neoplasm, malignancy, myeloma, paget, gout, infection, microcrystal, and radiology . Articles without english abstracts were excluded . Besides, certain relevant rheumatological, orthopedic, and radiological books were also used . Fractures are frequent in the elderly and result mainly from the effects of falls and osteoporosis . Low - impact falls, even from standing height, are the most common cause of injury in geriatric patients . Falling is a multi - factorial problem due to both extrinsic (e.g., environmental and housing conditions) and intrinsic risk factors (e.g., impaired mobility, loss of muscular strength, poor visual acuity, and medication, for instance corticoids). [46] osteoporosis, which is characterized by qualitatively normal, but quantitatively deficient bone, leads to bone fragility and increased risk of fractures . The general prevalence of osteoporosis in women is approximately 50% at the age of 85 years, while in men the prevalence is about 20% at that age . Ethnic differences also exist: mineral bone density is higher in black women and lower in asian women, while in white women evidence shows an intermediate value . Due to its precision, the most widely used quantitative technique is dual energy x - ray absorptiometry, which makes it possible to diagnose osteoporosis early, predict the risk of fracture, determine therapeutic intervention, and monitor response to treatment . This method grades bone loss according to standard deviations (sd) from the mean young adult value . A value less than 2.5 times the sd below the young adult mean, is considered to indicate osteoporosis . The main radiographic features of osteoporosis are increased radiolucency and cortical thinning, mainly in the spine, giving rise to a well - demarcated outline of the vertebral body, which has been described as, picture framing. Besides, in the spine, an increased biconcavity of the vertebral end plates, with a protrusion of the intervertebral disk into the vertebral body can be observed . However, early radiographic changes are subtle, as a bone loss of approximately 30% must occur before it can be detected . Vertebral compression fractures are the most common osteoporotic fractures, with a greater incidence in women over 60 years of age . They can be observed in up to 25% of women over 70 years of age and 40% of those over 80 years . After one episode, the risk of subsequent vertebral fracture increases at least four - fold, with substantially increased rates during the first year . These fractures are associated with higher mortality and significant morbidity, even though they are afforded little clinical attention . They often appear as multiple fractures with anterior wedging, and may be associated with significant deformity, leading to kyphosis . They are usually well demonstrated on radiographs, but can be better assessed with computed tomography (ct) or magnetic resonance (mr) imaging, when necessary . The latter imaging modality may be indicated in case of associated neurological symptoms a rare event or when a vertebroplasty is discussed, in order to better localize the recently fractured vertebra . Vertebral compression fractures in the elderly may involve several problems: first is differentiating between osteoporotic and malignant vertebral collapse (vc), particularly metastatic vc, as the latter is by far the most common malignant tumor affecting the skeleton in the elderly . In most patients, multiple, relatively symmetrical thoracolumbar vcs, associated with diffuse increased radiolucency are suggestive of osteoporosis [figure 1a]. The whole vertebral endplate is impacted, sometimes in association with increased or decreased radiolucency of the subjacent trabecular bone (band - like distribution of the abnormalities under the fractured vertebral end plate). Gas can also be seen under the fracture and is nearly pathognomonic of the benign nature of the collapse . There is no cortical osteolysis, although fractures can be identified with retropulsion of a bone fragment . Conversely, the following are a cause for concern: a single vc, involvement of the cervical spine, heterogeneous increased or decreased radiolucency that cannot be explained by an underlying end plate fracture, a focal collapse (on the ap or lateral view), or a bulging of the vertebral body or cortical osteolysis [figure 1b]. If in doubt or for a better assessment of the spinal canal and soft tissue, ct, or better yet, an mri can be performed, in order to differentiate between these two disorders . In benign fractures, an mri may show a band distribution of abnormal signal intensities on t1 and t2 weighted images, spared normal bone marrow signal intensity, retropulsion of a posterior bone fragment, and multiple compression fractures; on the contrary, signs such as a convex posterior border of the vertebral body, abnormal signal intensity of the posterior elements, an epidural mass or a focal paraspinal mass are suggestive of spinal metastasis. [1921] second, vertebral fractures can be missed following moderate trauma, because the fracture may be difficult to identify in an osteoporotic radiolucent bone and / or because the pain is attributed to another painful condition, such as degenerative changes . This is observed in particular in the cervical spine, particularly at c2, and may lead to secondary neurological complications [figure 2]. Ankylosis may be related to ankylosing spondylitis and diffuse idiopathic skeletal hyperostosis, which especially affect males and they have an estimated frequency of 5 to 15% in the elderly. [2225] these ankylosed spines become increasingly susceptible to injury, even in the event of low - energy impacts, and are prone to unstable vertebral fractures with an increased frequency of neurological complications, either primary or secondary, after unprotected transfers and manipulation . These lesions may be missed on plain films when non - displaced or mildly displaced, especially in hyperextension, whereas, they are well demonstrated on ct or mr imaging . However, the mri features can sometimes be misleading and mimic infectious spondylitis when signal abnormalities of the disco - vertebral junction are present . Caution is required when an elderly patient presents with an ankylosed spine, minor trauma or acute pain, especially if signal abnormalities of the anterior and posterior spine are observed [figure 3]. Usual benign vertebral compression (a) multiple levels, diffuse radiolucency, and whole vertebral endplate involvement (arrows); usual malignant vertebral compression characteristics (b) single vertebral involvement, focal and bulging of the posterior wall (arrowheads). Missed fracture of the odontoid process and vertebral body of c2 (arrows) in a patient with a history of fall one month before . Transverse fracture of thoracic vertebral body (arrow) and posterior arch (arrowhead) with posterior epidural hematoma (curved arrow) in an ankylosed spine depicted on (a) t1- and (b) t2-weighted sequences . Other important sites of fractures are the hip and the pelvis, which are associated with increased mortality and specific diagnostic problems in the elderly population . They are common and frequently result from lateral falls directly on the greater trochanter, in the case of hip fractures, and forward or backward falls in the case of pelvic fractures . Most fractures are easily diagnosed by conventional radiography, particularly fractures of the proximal femur . However, non - displaced fractures may be negative or equivocal, particularly if the bone is osteoporotic . If a clinical suspicion of a fracture persists, ct or better still mr imaging is advocated, as this modality can show the fracture line surrounded by bone marrow edema . They may occur everywhere, but are particularly common in the hip and pelvis, the majority being located in the sacrum and pubic ramus [figure 4]. Radiographic findings are often subtle or unremarkable, and in suspected cases mr imaging is indicated due to its superior accuracy as compared to other radiological methods . All the images should be carefully analyzed, as multiple insufficiency fractures are frequent [figure 5]. Subchondral fractures are particularly difficult to identify on radiographs, but the thin hypointense line that represents the fracture, surrounded by a variable degree of edema, is well demonstrated on an mri [figure 6]. This kind of fracture can be complicated by a necrosis of the subchondral bone, which is hypointense on t2 weighted images, and is not enhanced following gadolinium administration . Insufficiency fractures (arrows) located in the sacrum with adjacent edema (arrowheads), better depicted on (a) t1- and (b) t2- weighted sequences, respectively . Multiple fractures (white arrows) in (a) the proximal femur and (b) pubic ramus on coronal t1-weighted images subchondral fracture of the femoral head: (a) conventional radiography and (b) fat saturated sagittal t2-weighted image (white arrow). Osteoarthritis (oa) can be defined as a group of distinct, but overlapping diseases, which may have different etiologies, but similar biological, morphological, and clinical outcomes affecting the articular cartilage, subchondral bone, ligaments, joint capsule, synovial membrane, and periarticular muscles . Pathological changes can include fibrillation of cartilage, disruption of collagen fibers, changes in proteoglycan staining, chondrocyte proliferation, necrosis, and neovascularization . Oa is the most common joint disease in persons 65 years of age and above, with a radiographic prevalence as high as approximately 90% in women and 80% in men. [3335] its etiology is not fully understood, although there are several related factors, such as female gender, genetics, metabolism, and excessive mechanical stress . It frequently leads to decreased function and loss of independence . Although the joints of the hand are the most commonly affected, they are less likely to be symptomatic than the knee or hip [figure 7]. The diagnosis of oa is primarily based on clinical history and physical examination . Plain radiographs can help confirm both the diagnosis and grade the severity of the condition . The cardinal radiographic features of oa are focal / non - uniform narrowing of the joint space in the areas subjected to the most pressure, subchondral cysts, subchondral sclerosis, and osteophytes . However, the severity of the spondylolisthesis of the radiographic changes does not correlate with the clinical symptoms . Shoulder oa and rotator cuff disease are also fairly common in the elderly and are associated with significant shoulder pain and disability related to decreased shoulder movement . Direct signs of oa and indirect signs of large rotator cuff tears can be seen on plain films, including superior humeral head migration and remodeling of the acromion and the greater tuberosity . Ultrasound is another valuable tool for easy assessment of the rotator cuff tendons due to its good sensitivity and specificity [figure 8]. However, when surgical treatment is considered, arthro - ct, or mri may be preferred, in order to improve its planning . The lumbar and cervical spine is also frequently affected, with degenerative changes of the disk spaces, facet joints, and spinous processes, and sometimes with progressive scoliosis . Although the intervertebral disks are not synovial joints, the pathological changes are similar to those seen in the articular cartilage . Ct and mri are routinely used for the evaluation of narrowing of the central canal, lateral recesses, and neural foramina, when such an assessment is required (percutaneous or surgical treatment). Besides, mri can be useful in evaluating marrow changes in the vertebral plate, especially inflammatory changes (type i modic), which present a positive correlation with low back pain . Typical osteoarthritis of (a) the distal and proximal interphalangeal joints and (b) hip joint: joint space narrowing (arrows), subchondral cysts (arrowheads), osteophytes (curved arrows), and subchondral sclerosis (thick arrow). Ultrasound images in (a) the long and (b) short axis of the supraspinatus tendon demonstrating a complete tear (arrowheads). The frequency of microcrystal disorders also increases with age; this is mainly true of gout and calcium pyrophosphate dihydrate deposition (cpdd) arthropathy . Gout is the most common inflammatory arthritis in the elderly and is characterized by a disturbance of purine metabolism, with deposits in the joints, cartilage, and kidneys . The first metatarsophalangeal joint is the most common site of involvement in gouty arthritis [figure 9], but any joint can be involved . The typical radiographic features include eccentric nodular soft tissue masses (particularly suggestive of tophus when they are dense), close erosions (also suggestive when located at a distance from the joint and when associated with an elevated bony margin), preservation of the joint space, exuberant bony proliferation, and lack of periarticular osteoporosis . When the clinical, biological, and radiographic findings are uncertain, an ultrasound may be particularly helpful, as this technique may demonstrate more tophi [figure 10] and erosions than plain films, more synovitis than a clinical examination, suggestive hyperechoic aggregates in the synovium or in the joint fluid, which may be aspirated, and the double contour sign (an irregular hyperechoic band over the superficial margin of the articular cartilage, related to crystal deposition). [4244] calcium pyrophosphate dihydrate deposition (cpdd) disease is characterized by articular and periarticular tissue deposits (hyaline cartilage, fibrocartilage, and other soft - tissue structures), even in the spine [figure 11]. Unlike gout, in which the increase in serum urates leads to supersaturation and deposits in the joints, in cpdd, the calcium deposits usually appear in the cartilage in the absence of any serum abnormality . Even though such deposits may be seen in asymptomatic patients, they may be associated with the development of severe arthropathies or with acute pain related to the migration of the crystals in the synovial fluid, mimicking septic arthritis . Gout of the first mtp joint in conventional radiography: excentric dense nodular soft tissue mass (arrows), large erosions (arrowhead), lack of periarticular osteoporosis, and exuberant bony proliferation (curved arrow). Gout involvement of the mtp (a) shows the tophus (arrow) with hyperechogenic microcrystal deposits (arrowheads) and a thickened synovium (curved arrows). Another patient with tophus deposition in the dorsal midfoot (b) demonstrates microcrystal deposits (arrowhead) with acoustic shadows (thick arrows). Calcium pyrophosphate dihydrate deposition disease demonstrated by deposits of microcrystals in the menisci (arrow), articular cartilage (arrowhead), and periarticular soft tissue (curved arrow). Elderly patients are more prone to infection because of an increased incidence of predisposing disorders (diabetes mellitus, peripheral vascular disease, poor dentition,), immunosuppression and surgical procedures in this population (dental extractions, open heart surgery, prosthetic joint replacement,). The infectious agents affecting elderly patients are similar to those found in the younger population; although in the elderly, evidence shows an increased incidence of infection by nosocomial organisms (due to institutionalization and hospitalization). These infections can have a relatively benign course, as in the case of cellulitis, or serious consequences, as with necrotizing fasciitis . In patients over 80 years of age, the knee, shoulder, and hip are the most frequently affected joints . Another peculiarity of the elderly, uncommon in adults, is acute hematogenous osteomyelitis that mainly affects the spine . Attention should, moreover, be paid to the diagnosis of tuberculosis, as early manifestations are subtle and advanced disease mimics other infections, granulomatous diseases, and malignancy . Tuberculosis may affect the spine (about 50% of cases), with potential neurological consequences, or any joint, causing deformity [figure 12]. It should be noted that elderly men may present with bone lesions, as a manifestation of reactivation of the disease . As there are no pathognomonic imaging signs of musculoskeletal tuberculosis, the most conclusive means of reaching a diagnosis is by biopsy and culture, as chest radiographs and skin tests may not be positive in the elderly . As regards imaging techniques, magnetic resonance imaging is the modality of choice for musculoskeletal infection, as it best delineates the extension of soft tissue infections . Computed tomography, ultrasound, radiography, and nuclear medicine studies are considered to be ancillary . In some circumstances ultrasound can be very useful, as it allows the aspiration of fluid collected in the joints or soft tissue, the evaluation of structures adjacent to orthopedic hardware, or the assessment of small peripheral joints . Besides, in the case of debilitated elderly patients, a mobile ultrasound device can be easily moved close to their beds . (a) on conventional radiography there is mild osteoporosis, extensive soft tissue swelling, and large marginal erosion (arrowhead), (b) on doppler ultrasound the synovitis (arrows) and bone erosion (arrowhead) are well - depicted . Paget's disease is a bone disorder characterized by an increase in osteoclast - mediated bone resorption accompanied by osteoblast - mediated formation of new bone of inferior quality . The frequency of this condition is decreasing, but it still exists in 1 5% of persons over 50 years of age, varying according to geographic areas. [5356] the radiographic signs include an advancing wedge of bone resorption, an accentuation and coarsening of the bone trabeculae along lines of stress, cortical thickening, and enlargement of the bone [figure 13]. As a result, secondary osteoarthritis, insufficiency fractures, bowing of the bones, and even spinal cord or nerve root compression can be observed . In a minority of cases, sarcomatous degeneration may develop, mainly in the humerus, pelvis, and proximal femur, with a frequency believed to range from 0.9 to 2% . Paget's disease affecting the iliac bone demonstrated with coarsening of the bone trabeculae and cortical thickening (arrows). Tumors: tumors can be revealed by bone pain, pathological fractures, or disability, or be discovered as an incidental finding . Primary bone tumors in elderly patients are not common, but, due to the risk of metastases, any newly discovered bone lesion or one developing within a known pre - existing lesion must be assumed to be malignant until proven otherwise . Metastases: bone metastases are a common complication of malignant disease, especially of the breast, prostate, and lung . Although they are often multiple, a solitary metastasis is still more common than a primary neoplasm . Plain films are relatively insensitive for the detection of bone metastases, especially subtle lesions . Mr imaging is superior to ct in the detection of malignant marrow infiltration, and it has a better contrast resolution for visualizing soft tissue and spinal cord lesions . Even with the advances in mri, especially for bone marrow screening, bone scintigraphy continues to be used as an effective method for initial evaluation of the whole body for bone metastases . Sarcomas: in the elderly population, many sarcomas are secondary to a pre - existing disorder of the bone, such as paget's disease, or lesions treated by radiation . Chondrosarcoma must also be kept in mind, especially when isolated osteolytic lesions of the pelvis are seen, as this is the most common primary sarcoma of bone in adults . The cartilaginous nature of the lesion may be suggested by the presence of calcifications or by a lobulated hyperintense lesion in an mri [figure 14]. Chondrosarcoma of the pelvis in (a) conventional radiography appears as an isolated osteolytic lesion (arrows), (b) coronal t2-weighted image demonstrates a lobulated hyperintense lesion (arrowheads). Myeloma: myeloma is a characteristic disease in the elderly population, with a peak incidence in the eighth decade that results from an unregulated, progressive proliferation of neoplastic monoclonal plasma cells that accumulate in the bone marrow, leading to anemia and marrow failure . Osteolytic lesions may be present on plain films, but differentiating osteoporotic vertebral compression fractures from those associated with myeloma is a common diagnostic dilemma, as bone loss may be the first sign revealing the latter disease . Indeed, widespread osteoporosis, due to cytokine - mediated osteoclast activation, is common in patients with myeloma . This possible association must be kept in mind, particularly if other features are present, including unexplained back or bone pain . We used the following terms and some of its combinations: elderly, aged, epidemiology, fracture, osteoporosis, vertebral, hip, pelvis, arthritis, neoplasm, malignancy, myeloma, paget, gout, infection, microcrystal, and radiology . Articles without english abstracts were excluded . Besides, certain relevant rheumatological, orthopedic, and radiological books were also used . Fractures are frequent in the elderly and result mainly from the effects of falls and osteoporosis . Low - impact falls, even from standing height, are the most common cause of injury in geriatric patients . Falling is a multi - factorial problem due to both extrinsic (e.g., environmental and housing conditions) and intrinsic risk factors (e.g., impaired mobility, loss of muscular strength, poor visual acuity, and medication, for instance corticoids). [46] osteoporosis, which is characterized by qualitatively normal, but quantitatively deficient bone, leads to bone fragility and increased risk of fractures . The general prevalence of osteoporosis in women is approximately 50% at the age of 85 years, while in men the prevalence is about 20% at that age . Ethnic differences also exist: mineral bone density is higher in black women and lower in asian women, while in white women evidence shows an intermediate value . Due to its precision, the most widely used quantitative technique is dual energy x - ray absorptiometry, which makes it possible to diagnose osteoporosis early, predict the risk of fracture, determine therapeutic intervention, and monitor response to treatment . This method grades bone loss according to standard deviations (sd) from the mean young adult value . A value less than 2.5 times the sd below the young adult mean, is considered to indicate osteoporosis . The main radiographic features of osteoporosis are increased radiolucency and cortical thinning, mainly in the spine, giving rise to a well - demarcated outline of the vertebral body, which has been described as, picture framing. Besides, in the spine, an increased biconcavity of the vertebral end plates, with a protrusion of the intervertebral disk into the vertebral body can be observed . However, early radiographic changes are subtle, as a bone loss of approximately 30% must occur before it can be detected . Vertebral compression fractures are the most common osteoporotic fractures, with a greater incidence in women over 60 years of age . They can be observed in up to 25% of women over 70 years of age and 40% of those over 80 years . After one episode, the risk of subsequent vertebral fracture increases at least four - fold, with substantially increased rates during the first year . These fractures are associated with higher mortality and significant morbidity, even though they are afforded little clinical attention . They often appear as multiple fractures with anterior wedging, and may be associated with significant deformity, leading to kyphosis . They are usually well demonstrated on radiographs, but can be better assessed with computed tomography (ct) or magnetic resonance (mr) imaging, when necessary . The latter imaging modality may be indicated in case of associated neurological symptoms a rare event or when a vertebroplasty is discussed, in order to better localize the recently fractured vertebra . Vertebral compression fractures in the elderly may involve several problems: first is differentiating between osteoporotic and malignant vertebral collapse (vc), particularly metastatic vc, as the latter is by far the most common malignant tumor affecting the skeleton in the elderly . In most patients, multiple, relatively symmetrical thoracolumbar vcs, associated with diffuse increased radiolucency are suggestive of osteoporosis [figure 1a]. The whole vertebral endplate is impacted, sometimes in association with increased or decreased radiolucency of the subjacent trabecular bone (band - like distribution of the abnormalities under the fractured vertebral end plate). Gas can also be seen under the fracture and is nearly pathognomonic of the benign nature of the collapse . There is no cortical osteolysis, although fractures can be identified with retropulsion of a bone fragment . Conversely, the following are a cause for concern: a single vc, involvement of the cervical spine, heterogeneous increased or decreased radiolucency that cannot be explained by an underlying end plate fracture, a focal collapse (on the ap or lateral view), or a bulging of the vertebral body or cortical osteolysis [figure 1b]. If in doubt or for a better assessment of the spinal canal and soft tissue, ct, or better yet, an mri can be performed, in order to differentiate between these two disorders . In benign fractures, an mri may show a band distribution of abnormal signal intensities on t1 and t2 weighted images, spared normal bone marrow signal intensity, retropulsion of a posterior bone fragment, and multiple compression fractures; on the contrary, signs such as a convex posterior border of the vertebral body, abnormal signal intensity of the posterior elements, an epidural mass or a focal paraspinal mass are suggestive of spinal metastasis. [1921] second, vertebral fractures can be missed following moderate trauma, because the fracture may be difficult to identify in an osteoporotic radiolucent bone and / or because the pain is attributed to another painful condition, such as degenerative changes . This is observed in particular in the cervical spine, particularly at c2, and may lead to secondary neurological complications [figure 2]. Ankylosis may be related to ankylosing spondylitis and diffuse idiopathic skeletal hyperostosis, which especially affect males and they have an estimated frequency of 5 to 15% in the elderly. [2225] these ankylosed spines become increasingly susceptible to injury, even in the event of low - energy impacts, and are prone to unstable vertebral fractures with an increased frequency of neurological complications, either primary or secondary, after unprotected transfers and manipulation . These lesions may be missed on plain films when non - displaced or mildly displaced, especially in hyperextension, whereas, they are well demonstrated on ct or mr imaging . However, the mri features can sometimes be misleading and mimic infectious spondylitis when signal abnormalities of the disco - vertebral junction are present . Caution is required when an elderly patient presents with an ankylosed spine, minor trauma or acute pain, especially if signal abnormalities of the anterior and posterior spine are observed [figure 3]. Usual benign vertebral compression (a) multiple levels, diffuse radiolucency, and whole vertebral endplate involvement (arrows); usual malignant vertebral compression characteristics (b) single vertebral involvement, focal and bulging of the posterior wall (arrowheads). Missed fracture of the odontoid process and vertebral body of c2 (arrows) in a patient with a history of fall one month before . Transverse fracture of thoracic vertebral body (arrow) and posterior arch (arrowhead) with posterior epidural hematoma (curved arrow) in an ankylosed spine depicted on (a) t1- and (b) t2-weighted sequences . Other important sites of fractures are the hip and the pelvis, which are associated with increased mortality and specific diagnostic problems in the elderly population . They are common and frequently result from lateral falls directly on the greater trochanter, in the case of hip fractures, and forward or backward falls in the case of pelvic fractures . Most fractures are easily diagnosed by conventional radiography, particularly fractures of the proximal femur . However, non - displaced fractures may be negative or equivocal, particularly if the bone is osteoporotic . If a clinical suspicion of a fracture persists, ct or better still mr imaging is advocated, as this modality can show the fracture line surrounded by bone marrow edema . They may occur everywhere, but are particularly common in the hip and pelvis, the majority being located in the sacrum and pubic ramus [figure 4]. Radiographic findings are often subtle or unremarkable, and in suspected cases mr imaging is indicated due to its superior accuracy as compared to other radiological methods . All the images should be carefully analyzed, as multiple insufficiency fractures are frequent [figure 5]. Subchondral fractures are particularly difficult to identify on radiographs, but the thin hypointense line that represents the fracture, surrounded by a variable degree of edema, is well demonstrated on an mri [figure 6]. This kind of fracture can be complicated by a necrosis of the subchondral bone, which is hypointense on t2 weighted images, and is not enhanced following gadolinium administration . Insufficiency fractures (arrows) located in the sacrum with adjacent edema (arrowheads), better depicted on (a) t1- and (b) t2- weighted sequences, respectively . Multiple fractures (white arrows) in (a) the proximal femur and (b) pubic ramus on coronal t1-weighted images subchondral fracture of the femoral head: (a) conventional radiography and (b) fat saturated sagittal t2-weighted image (white arrow). Osteoarthritis (oa) can be defined as a group of distinct, but overlapping diseases, which may have different etiologies, but similar biological, morphological, and clinical outcomes affecting the articular cartilage, subchondral bone, ligaments, joint capsule, synovial membrane, and periarticular muscles . Pathological changes can include fibrillation of cartilage, disruption of collagen fibers, changes in proteoglycan staining, chondrocyte proliferation, necrosis, and neovascularization . Oa is the most common joint disease in persons 65 years of age and above, with a radiographic prevalence as high as approximately 90% in women and 80% in men. [3335] its etiology is not fully understood, although there are several related factors, such as female gender, genetics, metabolism, and excessive mechanical stress . Although the joints of the hand are the most commonly affected, they are less likely to be symptomatic than the knee or hip [figure 7]. The diagnosis of oa is primarily based on clinical history and physical examination . Plain radiographs can help confirm both the diagnosis and grade the severity of the condition . The cardinal radiographic features of oa are focal / non - uniform narrowing of the joint space in the areas subjected to the most pressure, subchondral cysts, subchondral sclerosis, and osteophytes . However, the severity of the spondylolisthesis of the radiographic changes does not correlate with the clinical symptoms . Shoulder oa and rotator cuff disease are also fairly common in the elderly and are associated with significant shoulder pain and disability related to decreased shoulder movement . Direct signs of oa and indirect signs of large rotator cuff tears can be seen on plain films, including superior humeral head migration and remodeling of the acromion and the greater tuberosity . Ultrasound is another valuable tool for easy assessment of the rotator cuff tendons due to its good sensitivity and specificity [figure 8]. However, when surgical treatment is considered, arthro - ct, or mri may be preferred, in order to improve its planning . The lumbar and cervical spine is also frequently affected, with degenerative changes of the disk spaces, facet joints, and spinous processes, and sometimes with progressive scoliosis . Although the intervertebral disks are not synovial joints, the pathological changes are similar to those seen in the articular cartilage . Ct and mri are routinely used for the evaluation of narrowing of the central canal, lateral recesses, and neural foramina, when such an assessment is required (percutaneous or surgical treatment). Besides, mri can be useful in evaluating marrow changes in the vertebral plate, especially inflammatory changes (type i modic), which present a positive correlation with low back pain . Typical osteoarthritis of (a) the distal and proximal interphalangeal joints and (b) hip joint: joint space narrowing (arrows), subchondral cysts (arrowheads), osteophytes (curved arrows), and subchondral sclerosis (thick arrow). Ultrasound images in (a) the long and (b) short axis of the supraspinatus tendon demonstrating a complete tear (arrowheads). The frequency of microcrystal disorders also increases with age; this is mainly true of gout and calcium pyrophosphate dihydrate deposition (cpdd) arthropathy . Gout is the most common inflammatory arthritis in the elderly and is characterized by a disturbance of purine metabolism, with deposits in the joints, cartilage, and kidneys . The first metatarsophalangeal joint is the most common site of involvement in gouty arthritis [figure 9], but any joint can be involved . The typical radiographic features include eccentric nodular soft tissue masses (particularly suggestive of tophus when they are dense), close erosions (also suggestive when located at a distance from the joint and when associated with an elevated bony margin), preservation of the joint space, exuberant bony proliferation, and lack of periarticular osteoporosis . When the clinical, biological, and radiographic findings are uncertain, an ultrasound may be particularly helpful, as this technique may demonstrate more tophi [figure 10] and erosions than plain films, more synovitis than a clinical examination, suggestive hyperechoic aggregates in the synovium or in the joint fluid, which may be aspirated, and the double contour sign (an irregular hyperechoic band over the superficial margin of the articular cartilage, related to crystal deposition). [4244] calcium pyrophosphate dihydrate deposition (cpdd) disease is characterized by articular and periarticular tissue deposits (hyaline cartilage, fibrocartilage, and other soft - tissue structures), even in the spine [figure 11]. Unlike gout, in which the increase in serum urates leads to supersaturation and deposits in the joints, in cpdd, the calcium deposits usually appear in the cartilage in the absence of any serum abnormality . Even though such deposits may be seen in asymptomatic patients, they may be associated with the development of severe arthropathies or with acute pain related to the migration of the crystals in the synovial fluid, mimicking septic arthritis . Gout of the first mtp joint in conventional radiography: excentric dense nodular soft tissue mass (arrows), large erosions (arrowhead), lack of periarticular osteoporosis, and exuberant bony proliferation (curved arrow). Gout involvement of the mtp (a) shows the tophus (arrow) with hyperechogenic microcrystal deposits (arrowheads) and a thickened synovium (curved arrows). Another patient with tophus deposition in the dorsal midfoot (b) demonstrates microcrystal deposits (arrowhead) with acoustic shadows (thick arrows). Calcium pyrophosphate dihydrate deposition disease demonstrated by deposits of microcrystals in the menisci (arrow), articular cartilage (arrowhead), and periarticular soft tissue (curved arrow). Elderly patients are more prone to infection because of an increased incidence of predisposing disorders (diabetes mellitus, peripheral vascular disease, poor dentition,), immunosuppression and surgical procedures in this population (dental extractions, open heart surgery, prosthetic joint replacement,). The infectious agents affecting elderly patients are similar to those found in the younger population; although in the elderly, evidence shows an increased incidence of infection by nosocomial organisms (due to institutionalization and hospitalization). These infections can have a relatively benign course, as in the case of cellulitis, or serious consequences, as with necrotizing fasciitis . In patients over 80 years of age, another peculiarity of the elderly, uncommon in adults, is acute hematogenous osteomyelitis that mainly affects the spine . Attention should, moreover, be paid to the diagnosis of tuberculosis, as early manifestations are subtle and advanced disease mimics other infections, granulomatous diseases, and malignancy . Tuberculosis may affect the spine (about 50% of cases), with potential neurological consequences, or any joint, causing deformity [figure 12]. It should be noted that elderly men may present with bone lesions, as a manifestation of reactivation of the disease . As there are no pathognomonic imaging signs of musculoskeletal tuberculosis, the most conclusive means of reaching a diagnosis is by biopsy and culture, as chest radiographs and skin tests may not be positive in the elderly . As regards imaging techniques, magnetic resonance imaging is the modality of choice for musculoskeletal infection, as it best delineates the extension of soft tissue infections . Computed tomography, ultrasound, radiography, and nuclear medicine studies are considered to be ancillary . In some circumstances ultrasound can be very useful, as it allows the aspiration of fluid collected in the joints or soft tissue, the evaluation of structures adjacent to orthopedic hardware, or the assessment of small peripheral joints . Besides, in the case of debilitated elderly patients, a mobile ultrasound device can be easily moved close to their beds . (a) on conventional radiography there is mild osteoporosis, extensive soft tissue swelling, and large marginal erosion (arrowhead), (b) on doppler ultrasound the synovitis (arrows) and bone erosion (arrowhead) are well - depicted . Paget's disease is a bone disorder characterized by an increase in osteoclast - mediated bone resorption accompanied by osteoblast - mediated formation of new bone of inferior quality . The frequency of this condition is decreasing, but it still exists in 1 5% of persons over 50 years of age, varying according to geographic areas. [5356] the radiographic signs include an advancing wedge of bone resorption, an accentuation and coarsening of the bone trabeculae along lines of stress, cortical thickening, and enlargement of the bone [figure 13]. As a result, secondary osteoarthritis, insufficiency fractures, bowing of the bones, and even spinal cord or nerve root compression can be observed . In a minority of cases, sarcomatous degeneration may develop, mainly in the humerus, pelvis, and proximal femur, with a frequency believed to range from 0.9 to 2% . Paget's disease affecting the iliac bone demonstrated with coarsening of the bone trabeculae and cortical thickening (arrows). Tumors: tumors can be revealed by bone pain, pathological fractures, or disability, or be discovered as an incidental finding . Primary bone tumors in elderly patients are not common, but, due to the risk of metastases, any newly discovered bone lesion or one developing within a known pre - existing lesion must be assumed to be malignant until proven otherwise . Metastases: bone metastases are a common complication of malignant disease, especially of the breast, prostate, and lung . Although they are often multiple, a solitary metastasis is still more common than a primary neoplasm . Plain films are relatively insensitive for the detection of bone metastases, especially subtle lesions . Mr imaging is superior to ct in the detection of malignant marrow infiltration, and it has a better contrast resolution for visualizing soft tissue and spinal cord lesions . Even with the advances in mri, especially for bone marrow screening, bone scintigraphy continues to be used as an effective method for initial evaluation of the whole body for bone metastases . Sarcomas: in the elderly population, many sarcomas are secondary to a pre - existing disorder of the bone, such as paget's disease, or lesions treated by radiation . Chondrosarcoma must also be kept in mind, especially when isolated osteolytic lesions of the pelvis are seen, as this is the most common primary sarcoma of bone in adults . The cartilaginous nature of the lesion may be suggested by the presence of calcifications or by a lobulated hyperintense lesion in an mri [figure 14]. Chondrosarcoma of the pelvis in (a) conventional radiography appears as an isolated osteolytic lesion (arrows), (b) coronal t2-weighted image demonstrates a lobulated hyperintense lesion (arrowheads). Myeloma: myeloma is a characteristic disease in the elderly population, with a peak incidence in the eighth decade that results from an unregulated, progressive proliferation of neoplastic monoclonal plasma cells that accumulate in the bone marrow, leading to anemia and marrow failure . Osteolytic lesions may be present on plain films, but differentiating osteoporotic vertebral compression fractures from those associated with myeloma is a common diagnostic dilemma, as bone loss may be the first sign revealing the latter disease . Indeed, widespread osteoporosis, due to cytokine - mediated osteoclast activation, is common in patients with myeloma . This possible association must be kept in mind, particularly if other features are present, including unexplained back or bone pain . Special attention is required in this population, as an early diagnosis can avoid delay in treatment, which is associated with increased morbidity and mortality . Besides, a better understanding of musculoskeletal diseases can lead to the implementation of effective preventive measures, thus reducing public health expenditure, and improving the quality of life in the elderly.

Migraine is the leading neurological cause for seeking medical care, and is associated with significant disability in the sufferer . The greatest impact is on migraineurs with headaches on more days than not, a condition defined as chronic migraine (cm). Cm is defined as at least 15 days of headache per month in which at least eight of the days fulfill migraine criteria and/or are treated with specific migraine medications, in the absence of a diagnosis of medication overuse headache . Patients with cm often had a history of episodic migraine that began in adolescence or early adulthood, reporting a process of transformation marked by headaches that become more frequent over years . Among migraineurs, defining risk factors for cm, or for the progression of episodic migraine to cm, identified risk factors include medication overuse, obesity, sleep problems, and psychiatric comorbidity [712]. Studies in both community and tertiary settings have demonstrated an association between migraine and several psychiatric conditions [8, 9]. However, the frequency of psychiatric disorders in both setting has not been compared before in a single study . Furthermore, differences in methods of studies based in community or tertiary centers prevent appropriate comparison . Population studies fail to conduct face - to - face assessments, while clinic - based studies carry the potential for selection bias . Studies focusing on best methods to address this gap are of interest, and one strategy is to compare data obtained from the community with those from specialty care, where methods of collection have been virtually identical, and that was the scope of this study . We compared demographic data and psychiatric comorbidity in a sample of individuals with cm from the community with another from a tertiary care clinic . In light of the fact that patients suffering from migraine and comorbid psychiatric disorders are greater health - care service users, we hypothesized that the frequency of psychiatric disorders, notably depression, is higher in patients followed in tertiary care . Community data were gathered in capela nova, a city from the state of minas gerais, brazil . According to the 2000 brazilian census, its population was 2,066 inhabitants (1,631 over the age of 10 years). The present study is part of an observational, cross - sectional and population - based study conducted in two phases [14, 15]. Initially, community health workers from the family health program directly interviewed all inhabitants aged 10 years or older for headache symptoms in the previous year . The family health program works through family health - care teams which are composed of one physician, one nurse, one auxiliary nurse, and four to six community health workers, and are assigned to specific geographical areas with defined populations of 6001,000 families . Activities provided by family health - care teams take place at primary care facilities, in patients homes, and in the community . In the first phase of our study (screening phase), trained community health workers screened for the occurrence of headaches using the following question: have you had any headache episode over the last 12 months? Those who screened positive were asked about headache frequency in the past month, and those reporting 15 days or more of headache were offered in - person assessment by neurologists with expertise in headache medicine . Three neurologists independently examined participants, and headaches were diagnosed according to the second edition of the international classification of headache disorders . Subsequently, all individuals aged 18 years or older with cm were assessed for psychiatric comorbidities . Psychiatric assessment was performed by an experienced psychiatrist using the mini international neuropsychiatric interview (mini). The same investigators involved in the community - based assessment used the very same procedures to diagnose consecutive patients attended at a university - based headache center in the first half of 2006 . This center is located in belo horizonte and is the only headache clinic in the state of minas gerais, brazil . The study followed the guidance of the regulatory norms of the brazilian national health council (resolution 196/1996) which is in accordance with the helsinki declaration . The protocol and all forms were reviewed and approved by the local ethics research committee . Data were transferred to epi - info 2000 by a study coordinator and analyzed using spss 12.0 . The relative frequencies of psychiatric comorbidities were stratified by headache type, and confidence intervals were calculated . Discrete data were compared between groups using the chi - square test or the fisher test (when anticipated values were small). For continuous non - parametric variables, the mann community data were gathered in capela nova, a city from the state of minas gerais, brazil . According to the 2000 brazilian census, its population was 2,066 inhabitants (1,631 over the age of 10 years). The present study is part of an observational, cross - sectional and population - based study conducted in two phases [14, 15]. Initially, community health workers from the family health program directly interviewed all inhabitants aged 10 years or older for headache symptoms in the previous year . The family health program works through family health - care teams which are composed of one physician, one nurse, one auxiliary nurse, and four to six community health workers, and are assigned to specific geographical areas with defined populations of 6001,000 families . Activities provided by family health - care teams take place at primary care facilities, in patients homes, and in the community . In the first phase of our study (screening phase), trained community health workers screened for the occurrence of headaches using the following question: have you had any headache episode over the last 12 months? Those who screened positive were asked about headache frequency in the past month, and those reporting 15 days or more of headache were offered in - person assessment by neurologists with expertise in headache medicine . Three neurologists independently examined participants, and headaches were diagnosed according to the second edition of the international classification of headache disorders . Subsequently, all individuals aged 18 years or older with cm were assessed for psychiatric comorbidities . Psychiatric assessment was performed by an experienced psychiatrist using the mini international neuropsychiatric interview (mini). The same investigators involved in the community - based assessment used the very same procedures to diagnose consecutive patients attended at a university - based headache center in the first half of 2006 . This center is located in belo horizonte and is the only headache clinic in the state of minas gerais, brazil . The study followed the guidance of the regulatory norms of the brazilian national health council (resolution 196/1996) which is in accordance with the helsinki declaration . The protocol and all forms were reviewed and approved by the local ethics research committee . Data were transferred to epi - info 2000 by a study coordinator and analyzed using spss 12.0 . The relative frequencies of psychiatric comorbidities were stratified by headache type, and confidence intervals were calculated . Discrete data were compared between groups using the chi - square test or the fisher test (when anticipated values were small). For continuous non - parametric variables, the mann in the community of 1,605 interviewed inhabitants, 57 (3.6%) had headaches on at least 15 days for at least three consecutive months . 43 (75.4%) had cm and 41 consented to being assessed by the psychiatrist (95.3% participation rate). In the headache center, 43 patients had cm (n = 453, 9.5%) and all consented in participating in the psychiatric assessment . Sociodemographic profiles were similar between groups with the exception of the mean number of years of formal education, lower in the community relative to the headache center (table 1).table 1demographic characteristics in individuals with chronic migraine from the community and a clinic - based samplecommunity (n = 41)headache center (n = 43)p valuegender men7 (17.1%) 2 (4.7%) 0.09 * * women34 (82.9%) 41 (95.3%) education (years of study) 826 (78.8%) 5 (11.6%) <0.001 * 9115 (15.2%) 15 (34.9%) 122 (6.1%) 23 (53.5%) age mean (sd)41.2 (17.2)35.7 (12.6)0.19 * * * range13731863 * chi - square, * * fisher, * * * mann whitney demographic characteristics in individuals with chronic migraine from the community and a clinic - based sample * chi - square, * * fisher, * * * mann whitney among individuals from the community, 65.9% of cases were diagnosed with any current psychiatric disorder, relative to 83.7% in those from the headache center (p = 0.06). The relative frequencies of some specific diagnoses were remarkably high in both groups, despite not being statistically different . In the headache center, the most prevalent disorders were simple phobia (41.9%), generalized anxiety disorder (34.9%) and major depression (32.6%). In the community, the same disorders were also the most common ones: generalized anxiety disorder (39.0%), phobias (29.3%), and major depression (29.3%). Bipolar disorder was not seen in the community and was diagnosed in two cases from the headache center . The frequency of antidepressants use was similar in the headache center (51.2%) and in the community (44.4%) (p = 0.51). Table 2 summarizes these data.table 2current psychiatric comorbidities in individuals with chronic migraine from the community and a clinic - based sampleheadache center (n = 43)community (n = 41)p valueany diagnosis36 (83.7%) 27 (65.9%) 0.06*one or two psychiatric diagnoses21 (48.8%) 16 (39.0%) 0.36three or more psychiatric diagnoses15 (34.9%) 11 (26.9%) 0.42major depression14 (32.6%) 12 (29.3%) 0.74*dysthymia9 (20.9%) 9 (22.0%) 0.99**bipolar disorder2 (4.7%) 0 (0%) 0.23**generalized anxiety disorder15 (34.9%) 16 (39.0%) 0.69*specific phobia18 (41.9%) 12 (29.3%) 0.23*obsessive compulsive disorder9 (20.9%) 10 (24.4%) 0.70*somatization7 (16.3%) 3 (7.3%) 0.31**eating disorders2 (4.7%) 1 (2.5%) 0.99*alcohol abuse0 (0.0%) 2 (4.9%) 0.23 * * * chi - square, * * fisher current psychiatric comorbidities in individuals with chronic migraine from the community and a clinic - based sample * chi - square, * * fisher to the best of our knowledge this is the first study to compare the frequency of psychiatric comorbidity of cm in community and tertiary care clinic samples . The frequency of psychiatric disorders in cm was elevated in both settings, tending to be higher in the tertiary care sample . While psychiatric comorbidity in episodic migraine has been well established in the literature, psychiatric disorders have been less studied in cm . Only a few studies have addressed psychiatric comorbidities of cm in population - based samples, finding increased levels of depression and anxiety disorders even in comparison with episodic migraine patients [11, 20]. One limitation of these studies was the use of self - report questionnaires rather than clinical interview in ascertaining psychiatric diagnosis . In the present study we found that up to a third of our patients in each setting had depression . A similar rate was described in the american migraine prevalence and prevention (ampp) study, a population - based survey based on mailed questionnaires . In that study, depression was assessed by self - report of a physician diagnosis and by the patient health questionnaire (phq-9)depression module . One interesting result from the ampp study was that cm patients were twice as likely to have depression as assessed by phq-9 in comparison with episodic migraine patients [30.2 and 17.2% respectively; or (95% ci) = 2.0 (1.67 to 2.40), p <0.001]. Cm patients were also approximately twice as likely to report anxiety [30.2 vs. 18.8% respectively; or (95% ci) = 1.8 (1.51 to 2.15), p <0.001]. The international burden of migraine study (ibms) also found higher levels of anxiety and depression in cm when compared with episodic migraine . Regarding anxiety syndromes, generalized anxiety disorder and phobias seem to be comorbid with the migraine spectrum [2024]. Interestingly the frequency of obsessive compulsive disorder was significantly high (between 20 and 25%) in cm patients in comparison with its prevalence in the general population (2%). Only few previous studies have pointed out this association between obsessive compulsive disorder and migraine that may be associated with underlying serotonin system dysfunction . Bipolar disorder is also comorbid with migraine; migraineurs without aura are 2.4 times more likely to have bipolar disorder type 1, and the ratio increases to 7.3 when the diagnosis is migraine with aura [13, 22]. For bipolar disorder type 2, we failed to detect this association, likely because of small size of the samples . Drug abuse has not been traditionally associated with migraine [22, 24], but a recent study reported that illicit drug abuse may be more frequent in migraine patients with depression or post - traumatic stress disorder . Demographic profiles were similar in both groups, and the vast majority of individuals with cm were women . It must be highlighted that the assessed community was from a small city, while the patients at the headache center mainly came from a large urban center . Nonetheless, studies of migraine that have enrolled subjects with different demographic features also found striking similarities regarding the risk of psychiatric comorbidities, once more pointing to shared biological factors as a plausible mechanism for the comorbidity [22, 24]. Specific genotypes coding d2 dopaminergic receptors, dysfunction in tyramine conjugation, changes in the metabolism of serotonin and catecholamines and in estrogen levels have been considered to explain the comorbidities [2730]. It is worth mentioning, however, that we have comprehensively and systematically assessed almost all patients with cm from an entire population of a small city . We did not assess the differential disability associated with headache and psychiatric disorders in the individuals . Finally, our findings were not adjusted for other confounding factors, such as parameters of headache severity (intensity, duration associated symptoms), obesity, sleep disorders, use of psychotropic medication and household income . We partly justify these latter limitations by arguing that the demands on patients and resources in conducting these missing assessments could jeopardize the community assessment, since most interviews were conducted in participant households . In conclusion, the present study suggests that psychiatric comorbidity in cm is elevated in the community and clinical settings, tending to be more common in cm patients from a headache center.

M1dg (3-(2-deoxy--d - erythro - pentofuranosyl)-pyrimido[1,2-]purine-10(3h)-one) is one of many endogenous exocyclic lesions derived from the reaction of dna with bifunctional electrophiles generated by lipid, protein, or dna peroxidation . Being an endogenous constituent of human and rodent genomes, m1dg is mutagenic in bacterial and mammalian cells and leads to mispairing when replicated in vitro by multiple different dna polymerases . We have previously developed a monoclonal antibody against m1dg and incorporated the antibody into analytical schemes using immunoaffinity purification of m1dg followed by mass spectrometric detection . These studies indicated that adult humans excrete m1dg at a rate of 12 fmol / kgd in urine . Subsequent investigations into the metabolism and elimination of m1dg revealed that administration of m1dg to rodents at doses from 8 mg / kg to 6 pg / kg leads to its rapid disappearance from plasma due to oxidation and formation of the single, stable metabolite, 6-oxo - m1dg (3-(2-deoxy--d - erythro - pentofuranosyl)-pyrimido[1,2-]purine-6,10(3h,5h)-dione). In both humans and rats, xanthine oxidase appears to be responsible for this oxidation, while aldehyde oxidase also plays a role in m1dg metabolism in humans . Because 6-oxo - m1dg is the sole metabolite of m1dg in rats, it appears to be an ideal surrogate for m1dg as an in vivo biomarker for oxidative stress . All studies of m1dg metabolism in vivo have been conducted with exogenously administered deoxynucleoside . Thus, it is not known if 6-oxo - m1dg is actually present in unadulterated intact animals . In order to address this question, we developed a highly specific monoclonal antibody (mab) against 6-oxo - m1dg and covalently linked the antibody to sepharose beads . The resultant antibody - sepharose matrix (the gel) was used for the immunoaffinity purification of 6-oxo - m1dg from urine and feces followed by lc - ms / ms quantification against the stable isotope - labeled internal standard, [n5]-6-oxo - m1dg . Utilizing this method, we report for the first time that 6-oxo - m1dg is endogenously produced in rodents and is excreted in urine and feces . Na2co3, nahco3, nacl, water, acetonitrile, mariculture keyhole limpet hemocyanin (mcklh), hcl, acetic acid, and methanol were obtained from thermo fisher scientific (waltham, ma, usa). Abts (diammonium salt), thimerosal, kh2po4, na2hpo4, kcl, 30% h2o2, tween 20, bovine serum albumin (bsa), dimethylformamide (dmf), dimethylsulfoxide (dmso), k2co3, sodium periodate, sodium methoxide, nabh4, hypoxanthine, xanthosine, xanthine, ethyl cis-3-iodoacrylate, ethyl cis-3-bromoacrylate aminopterin, ethanolamine, cyanogen bromide - activated sepharose beads, diethylene glycol, 2-deoxyguanosine, sodium hydroxide, formic acid, sodium azide, glycine, and citric acid were obtained from sigma - aldrich (st . Louis, mo, usa). Horseradish peroxidase - conjugated goat antimouse igg (h + l) [0.8 mg / ml] and igg (fc) were purchased from jackson immunoresearch (west grove, pa, usa). Phosphate buffered saline (pbs) was purchased from invitrogen (carlsbad, ca, usa). The synthesis of 6-oxo - m1guo was based on a previously described synthetic scheme with some modifications . Guanosine was dissolved in a minimum amount of a dmf / dmso mixture (1:1, v / v). K2co3 (1.5 equiv) was added to the solution and the reaction mixture was held at 6065 c for 18 h. ethyl cis-3-iodoacrylate (0.25 equiv per h for 5 h) was added to the reaction mixture . The residue was dissolved into a minimum amount of methanol, and sodium methoxide (1.5 equiv) was added (dropwise in a 0.5 m solution). 6-oxo - m1guo was purified on biotage sp1 flash chromatography system (biotage, uppsala, sweden) using the following gradient; 1% to 5% acetonitrile in 30 column volumes followed by a 5% to 15% gradient in 10 column volumes . Separation was achieved on a 12 + m c18 column, and the purified compound showed a single sharp peak by reverse - phase hplc . H nmr (600 mhz, dmso - d6): 8.65 (d, 1h, h8, j = 7.8 hz), 8.29 (s, 1h, h2), 6.24 (d, 1h, h7, j = 8.4 hz), 5.82 (d, 1h, h1), 5.48 (d, 1h, h2oh), 5.20 (d, 1h, h3oh), 5.03 (d, 1h, h5oh), 4.49 (d, 1h, h2), 4.15 (d, 1h, h3), 3.91 (d, 1h, h4), 3.633.55 (m, 2h, h5). The h nmr may be seen in figure a of the supporting information . 6-oxo - m1dg and its stable isotope - labeled analogue, [n5]-6-oxo - m1dg, were synthesized as previously described . Briefly, anhydrous dg (1.6 g, 6 mmol) and anhydrous k2co3 (0.93 g, 6.75 mmol) were dissolved in anhydrous dmf (18 ml). One milliliter of a 0.55 m solution of ethyl cis-3-bromoacrylate in anhydrous dmf was added to the reaction every 15 min for 4 h. the reaction was stirred for an additional 2 h then cooled and filtered . The filtrate was evaporated under high vacuum and the residue was dissolved in water (4 ml) and 5% acetic acid (4 ml). The product was purified by reversed - phase (c18) medium pressure liquid chromatography with a biotage sp1 apparatus (biotage) using a prepacked 25 75 mm biotage flash 25 + s cartridge (kp - c18-hs) and a flash 25 c18 samplet for sample loading . A pure final product, 6-oxo - m1dg (0.92 g, 50.6%), was obtained . [n5]-6-oxo - m1dg was synthesized by substituting [n5]-dg for dg (cambridge isotope laboratories, andover, ma, usa) and employing the scheme described above . The h nmr of 6-oxo - m1dg and its stably labeled analogue matched the spectrum previously reported for this compound . Mass spectrometric analysis of 6-oxo - m1dg showed an [m + h] peak at m / z 320, while analysis of [n5]-6-oxo - m1dg showed an [m + h] peak at m / z 325 . The m / z = 325 corresponds to the 6-oxo - m1dg value plus 5 additional mass units from the 5 n atoms incorporated into the [n5]-dg starting material . A m / z 320 peak was not observed for [n5]-6-oxo - m1dg, indicating that no unlabeled 6-oxo - m1dg was present in the internal standard . 6-oxo - m1guo (12 mg) was dissolved in 750 l of 100 mm aqueous sodium periodate . Protein (20 mg of bsa or mcklh) was reconstituted with 700 l of pbs preadjusted to a ph of 9.5 with 5% k2co3 . The 6-oxo - m1guo solution and the protein after 1 h, 45 l of a 1 m diethylene glycol solution was added to the mixture (to quench excess oxidizing agent) followed by 700 l of 0.45 m nabh4 (aqueous). After another 12 h, the ph of the mixture was adjusted to 7.0 with 1.0 m formic acid . The mixture was kept at this ph for 1 h. then, the ph was increased to 8.5 by the careful addition of 1 m aqueous ammonium hydroxide solution . This mixture was dialyzed against pbs buffer twice for 24 h. the sample was lyophilized and stored at 4 c . Scheme 1 shows the conjugation reaction of 6-oxo - m1guo with a lysine residue of the carrier protein . Figure b of the supporting information depicts the scheme for preparation of the conjugated protein . Four balb / cj mice and four a / j mice (the jackson laboratory, bar harbor, me, usa) were injected subcutaneously with 50 g of 6-oxo - m1guo - klh and freund s complete adjuvant (primary boost). Four wk after the initial immunization, the mice were boosted subcutaneously (first boost) with the same dose of conjugate, but with the substitution of incomplete adjuvant (which was also used in subsequent boosts). Two wk after the first boost, the mice were tail bled and antibody titers were assessed by direct and competitive elisa as described below . A second boost was administered 4 wk subsequent to the first, and after 2 wk, sera were again extracted and subjected to elisa evaluation . A third boost was administered 25 wk after boost two, and the sera were collected and screened approximately 2 wk later . On the basis of the cumulative elisa data, a single balb / cj mouse (balb / cj r) showing the most selective and concentrated anti-6-oxo - m1dg titer was chosen for a fourth and final boost given intraperitoneally and lacking adjuvant to prepare it for splenocyte extraction . Four days after the final boost, the mouse was sacrificed by cervical dislocation and the spleen harvested . Splenocytes were isolated and subjected to polyethylene glycol - mediated fusion with both sp/20 and ns1 murine myeloma cells (obtained from the university of virginia and the university of nebraska medical univeristy school of dentistry, respectively) and allowed to recover for 24 h in liquid culture . The products of the fusion were evenly distributed into 24 96-well plates, with 12 plates corresponding to fusions of sp/20 cells and splenocytes and 12 plates corresponding to fusions of ns1 cells and splenocytes . Cells were grown in iscove s modified dulbecco s medium with 20% fetal bovine serum (invitrogen). Hybridomas were selected by growing in the presence of aminopterin (5 10 m, sigma) and ht supplement (1:100 dilution, invitrogen) for 14 days with media and aminopterin replenishment every 3 days . Supernatant was removed and screened for antigen - specific antibodies by elisa, with 6-oxo - m1dguo - bsa conjugate used as the antigen (described below). Positive hybridoma supernatants were rescreened in the presence of xanthine and 6-oxo - m1dg . Xanthine was employed as a competitor to identify antibodies not specific to 6-oxo - m1dg . Clones exhibiting anti-6-oxo - m1dg activity and specificity in addition to positive growth and productivity were selected and plated on 24-well plates . Of these, five clones were chosen based on activity, specificity, growth, and productivity as determined by direct and competitive elisa analysis (described below) and then subsequently subjected to two rounds of subcloning . The clones were subsequently cryopreserved . Under vanderbilt institutional care and use committee protocol #m-07 - 109, the vanderbilt antibody and protein resource core is permitted to use freund s complete adjuvant in the manner described above . The following solutions were prepared as follows and their use is described below: (a) carbonate - bicarbonate coating buffer (ph 9.6) was prepared from na2co3 (1.59 g / l), and thimerosal (0.10 g / l); (b) pbs - tween (ph 7.4) was prepared from nacl (8.00 g / l), tween 20 (1.00 ml / l), and thimerosal (0.10 g / l); (c) bsa (5.0 g) mixed in pbs - tween (500 ml); (d) 1 nm abts solution in 70 mm citrate - phosphate buffer (ph 4.2) was prepared from citric acid (5.64 elisa quality microtiter plates (thermo fisher scientific; 96-well immulon 2hb flat bottom microtiter plates or 384-well 4hb flat bottom plates) were coated with 50, 75, or 100 l of a 10 g / ml solution of 6-oxo - m1guo - bsa antigen in carbonate - bicarbonate coating buffer (a) and incubated at 4 c overnight . Plates were washed three times with 100 l of pbs - tween (b) using a bio - tek elx405 automatic microplate washer (winooski, vt, usa), then incubated with 100 l of pbs - tween (b) for 30 min at 37 c . Aliquots of murine serum dilutions, hybridoma supernatants, or purified antibody (depending on the stage of the antibody development process) were then incubated in the coated wells with or without the addition of varying concentrations of 6-oxo - m1dg and structural analogues (figure 1) in a final volume of 100 l pbs for 60 min at 37 c . For wells that included 6-oxo - m1dg or structural analogues, the serum dilution used was 1:5000 in pbs - tween (b), and the compounds depicted in figure 1 were preincubated in the dilute murine serum for 45 min prior to the serum addition step . This allowed the nonspecific antibodies to bind the structural analogues, theoretically leaving behind only 6-oxo - m1dg - specific antibodies . Initial analysis revealed that xanthine was recognized nearly as well as 6-oxo - m1dg, which led to the use of this structural analogue in further competition studies . 6-oxo - m1dg, m1dg, and structural analogues that were used as competitive antigens during elisa analysis of murine sera, hybridomal supernatant, and purified antibodies . The plates were then washed three times with pbs - tween (b). Aliquots of diluted horseradish peroxidase - conjugated goat antimouse igg fc region - specific or igg (h + l) secondary antibody (diluted 1:5000 in pbs - tween / bsa) were added, and the plates were incubated at 37 c for 60 min . After 60 min, the plates were washed three times and 100 l of a freshly prepared abts solution (1.8 l of h2o2 per 1 ml of abts) was immediately added to each well . To determine peroxidase activity, the absorbance at 414 nm was measured after 15 and 30 min for each well using a bio - tek powerwave ht 340 plate reader with gen5 software . Selected clones were scaled up, inoculated into one - liter bioreactors (wilson wolf manufacturing, new brighton, mn, usa) and grown for 34 wk . Purification of mab from the bioreactor supernatants was achieved by affinity chromatography on protein - g sepharose (ge healthcare, piscataway, nj, usa) followed by a final desalting step into pbs . Purified mab were isotyped and subsequently quantified by sds - page electrophoresis followed by infrared coomassie staining . Purified anti-6-oxo - m1dg mab was loaded onto pd-10 columns (ge healthcare) equilibrated with 25 ml coupling buffer (0.1 m nahco3, 0.5 m nacl, ph 8.0 in water). Cyanogen bromide - activated sepharose beads (3.75 g) were prepared for conjugation by rapid swelling in 1 mm hcl followed by filtering, rinsing with 400 ml of 1 mm hcl and then 10 ml of coupling buffer . The sepharose beads were transferred to the antibody solution and gently rotated at 4 c for about 20 h. the mixture was then filtered and washed with 500 ml of coupling buffer, and the residue was divided into four parts . Each quarter was placed in 14 ml of ethanolamine solution (1.0 m, ph 8.0) and gently rotated overnight at 4 c . The sepharose beads were then filtered and washed with a low ph acetate buffer (0.1 m acetic acid, 0.1 m naoh, and 0.5 m nacl) followed by a 0.1 m tris hcl buffer (0.5 m nacl, ph 8.0). Each wash used 75 ml of buffer, and the wash cycle was repeated two more times . The resulting residue was suspended in 20 ml of 0.1 m tris buffer with 0.5 m nacl and 0.2% nan3 (ph 8.0), and the final product (the gel) was stored at 4 c . The gel was used to recover 6-oxo - m1dg and [n5]- 6-oxo - m1dg from various fluids by the following general procedure . An aliquot of gel was prepared for use by rinsing with the following solutions: 50 ml of 0.1 m glycine (ph 2.7), 20 ml h20, 50 ml methanol, and 50 ml pbs . This cleaning procedure was necessary to remove any 6-oxo - m1dg that had bound to the mab during preparation . The ph of the fluid to be analyzed was adjusted to 7.08.0 with acetic acid or ammonium hydroxide . An aliquot of the washed and reconditioned gel was added to the sample, and the suspension was mixed gently for 14 h. the sample / gel suspension was filtered through a precleaned empty polypropylene spe cartridge fitted with a polypropylene frit (sigma - aldrich). The filtered gel was washed with 10 ml of pbs, and the analytes were eluted with 2 ml of methanol . The eluant was dried under n2 gas, capped, and stored at 20 c . The sample was reconstituted in 9:1 water / methanol (v / v) immediately prior to lc - ms / ms analysis . Urine and feces were collected at 24 h intervals and stored at 20 c following collection . Urine samples were analyzed by thawing, adding 200500 fmol of [n5]-6-oxo - m1dg, and centrifuging the samples at 4000 g for 25 min at 4 c . An aliquot of supernatant was diluted 1015-fold with pbs, and the ph of the final solution was adjusted to 7.5 0.5 with either acetic acid or sodium hydroxide . The gel (prepared as described above) was introduced after ph adjustment, and the suspension was rotated gently end - over - end for 14 h. the suspension was filtered and the analyte eluted from the gel as described above . The weighed sample was transferred to a clean vessel with 40 ml of pbs, and 200500 fmol of [n5]-6-oxo - m1dg were added . Dispersion of the sample was accomplished by rotating end - over - end for 23 h followed by sonication for 35 min . The suspension was then filtered through a mesh screen, with the collected solid material rinsed with 10 ml pbs . The supernatant was transferred to a clean vessel, diluted roughly 5 times with pbs, and the antibody gel (prepared as described above) was added . The suspension was rotated gently end - over - end for 14 h, filtered, and the analyte eluted from the gel as described above . Final lc - ms / ms analysis of gel - purified samples was accomplished on a thermo surveyor autosampler and ms pump in - line with a thermo quantum triple quadrupole mass spectrometer (thermo fisher scientific). The mass spectrometer was equipped with an electrospray source and operated in positive ion mode . 6-oxo - m1dg and its internal standard, [n5]-6-oxo - m1dg, were detected via selected reaction monitoring (srm) with the following transitions, respectively; m / z 320 204 and 325 209 . In both cases, the transition corresponds to cleavage of the glycosidic bond and loss of the deoxyribose moiety, with the positive charge remaining on the base . The analytes were chromatographed on the following reverse - phase gradient system; 2% b to 15% b in 5 min, 15% b to 80% b in 1.5 min, followed by a 1 min hold at 80% b. component a was water and component b was methanol / acetonitrile (3:1), and each component contained 0.1% acetic acid (v / v). The column used was a phenomenex c18 (phenomenex, torrence, ca, usa; 10.0 0.2 cm, 3 m) held at 3540 c . The column was equilibrated at the initial conditions for 3 min prior to each injection . 6-oxo - m1dg was quantitated by stable isotope dilution against [n5]-6-oxo - m1dg . Lc - ms / ms data was acquired and processed by xcalibur software (thermo fisher scientific). The overall scheme of the antibody generation and isolation process is depicted in figure 2 . Eight mice (four balb / cj and four a / j) were injected with 6-oxo - m1 g conjugated to klh as described in the materials and methods section . At 6, 10, and 35 wk after the initial innoculation, the mice were tail bled, and direct and competitive elisa analyses of the sera were performed (figure 2, step 1). All three bleeds from all 8 mice showed robust responses against the 6-oxo - m1g - bsa antigen, indicating that the immune systems of the mice were producing significant amounts of antibodies against the 6-oxo - m1 g portion of the innoculant . Competitive elisa screenings were executed to assess antibody specificity, with 6-oxo - m1dg and a series of structural analogues (figure 1) serving as competing antigens . Eight mice were inoculated with adduct / protein conjugate, followed by tail bleeding and testing for the presence of 6-oxo - m1dg antibodies using direct elisa analysis (step 1). Splenocytes from a single mouse, balb / cj r, were fused with myeloma cells, plated on twenty - four 96-well plates, and screened using direct and competitive elisa analyses (step 2). From this screen, five parental cell lines were chosen for subcloning and further screening (step 4). On the basis of these final screenings, the cell line 6c9ba4c12 was chosen for scale - up and antibody purification (step 5). Figure 3 depicts a competitive elisa analysis of the 35-week serum from mouse balb / cj r. this analysis employed 6-oxo - m1dg as the competing antigen and a robust concentration dependent decrease in optical density is observed, indicating the presence of anti-6-oxo - m1dg antibodies in the sera from this mouse . The results of similar analyses of the sera of all 8 inoculated mice can be seen in supporting information figure c. supporting information figure d displays the results from competitive elisa analyses of the 35-week serum from all 8 inoculated mice where m1dg is employed as the competing antigen . Increasing amounts of m1dg do not result in a decrease in optical density, indicating that m1dg does not bind to the anti-6-oxo - m1dg antibodie(s) present in the sera . 6-oxo - m1dg antibodies are present in the serum of mouse balb / cj r. the serum of mouse balb / cj r was subjected to competitive elisa analysis with 6-oxo - m1dg used as the competitor . The displayed results represent the average of spectroscopic readings at 15 and 30 min postaddition of abts substrate . On the basis of the elisa analysis of murine sera, a single mouse (balb / cj r) was chosen for hybridoma production . Splenocytes from balb / cj r were fused with both sp/20 and ns1 myeloma cells (figure 2, step 2), and 1670 hybridomas were formed from 2304 fusions wells . Elisa analysis revealed that of the 1670 hybridomas, 180 showed production of anti-6-oxo - m1dg antibodies . These were grown in 24-well plates (figure 2, step 3), and the supernatants were subjected to further elisa screenings . On the basis of the screening data, five hybridomas were selected for subcloning and further screenings (figure 2, step 4). Figure 4 shows a representative elisa analysis of the supernatants from two subclones (b and e2) of parental hybridoma cell line 6c9 in which xanthine and 6-oxo - m1dg are employed as competing antigens . The presence of unbound 6-oxo - m1dg decreases the optical density of both b and e2 supernatants, indicating the presence of anti-6-oxo - m1dg antibodies . For subclone e2, xanthine has a similar effect, demonstrating that e2 s mab are not specific to 6-oxo - m1dg . However, xanthine has no effect on the optical density of subclone b supernatant, suggesting that mab from subclone b are specific to 6-oxo - m1dg . On the basis of these data, subclone b was subjected to further subcloning but e2 was not . Supporting information figure e illustrates that cell line 6c9ba4, a subclone of hybridoma 6c9b, retained activity against 6-oxo - m1dg but did not react to xanthine . The specificity of antibodies produced by two hybridoma cell lines (b and e2) subcloned from the parental hybridoma cell line 6c9 were assessed using competitive elisa analysis . The displayed results represent the average of spectroscopic readings of subclones e2 and b at 15 and 30 min postaddition of abts substrate . On the basis of a review of the cumulative elisa data, a final daughter cell line was selected from each parental hybridoma line, and these daughter cell lines were cryopreserved . The elisa data indicated that antibodies from the cell line 6c9ba4c12 displayed the most promising expression and specificity . Thus, this cell line was subjected to large - scale expression and antibody purification . Figure 5 shows the elisa responses of the mab purified from 6c9ba4c12 in the presence of 6-oxo - m1dg and structural analogues . Only 6-oxo - m1dg attenuated the optical density, indicating a specificity of the purified mab for 6-oxo - m1dg over close structural analogues . Purified antibodies from the hybridomal subclone (6c9ba4c12) were screened for specificity for 6-oxo - m1dg in the presence of structural analogues using competitive elisa analyses . The purified anti-6-oxo - m1dg mab was covalently linked to sepharose beads as described above . The purification and analysis protocol outlined in materials and methods was used to quantify aliquots of 10 mm potassium phosphate (ph 8.0) spiked with varying amounts of 6-oxo - m1dg (0.25, 2.5, and 25 pmol) and 1.0 pmol of the internal standard, [n5]-6-oxo - m1dg (n = 3 for each concentration of 6-oxo - m1dg). As shown in table 1, the experimentally determined amount of 6-oxo - m1dg was within 12% of the known amount at each level . The average percent recovery of 6-oxo - m1dg ranged from 94% to 74%, while the internal standard was recovered at an average rate of 79% . Thus, the gel was able to bind 6-oxo - m1dg from an aqueous solution and released the analyte in the presence of methanol . The amount detected,% accuracy, and% recovery values are shown as the mean s.d . The limit of detection (lod) of the described lc - ms / ms system was established at approximately 10 fmol 6-oxo - m1dg on - column (supporting information figure f, top) by analyzing a series of increasingly dilute 6-oxo - m1dg solutions . Given this lod and the percent recovery observed from urine and fecal samples (approximately 30%), the limit of quantification of the assay was estimated to be 50 fmol per sample . Additionally, the lc - ms / ms system provided a linear response to 6-oxo - m1dg solutions over a 5000-fold concentration range and a single [n5]-6-oxo - m1dg concentration (supporting information f, bottom). These results indicate that the gel provided a sufficient level of recovery of the analyte and its internal standard as well as sufficient capacity for the expected levels of 6-oxo - m1dg present in rat urine and feces . They also demonstrate that the lc - ms / ms analysis of the purified sample provided sensitive and accurate quantification . Urine and feces collected from male sprague dawley rats over 3 days were analyzed as described in the materials and methods section . 6-oxo - m1dg was detected in urine from only one subject, animal 3, at a rate of 188 fmol / kgd . However, 6-oxo - m1dg was eliminated in feces in all the animals at a rate of 3501893 fmol / kgd . Thus, it appears fecal elimination is the main route by which that 6-oxo - m1dg is excreted from the body . Values are given as the mean amount observed in fmol / kgd s.d . (n = 35 for feces and 2 for urine). While the amounts of 6-oxo - m1dg are low, the assay provided a sufficient signal - to - noise ratio to permit accurate quantification . The upper trace (m / z 320 204) shows the analyte, while the bottom trace (m / z 325 209) represents the [n5]-6-oxo - m1dg internal standard . The inset of figure 6 is the internal standard alone after recovery from pbs . There is no peak in the 6-oxo - m1dg trace of the inset, which is illustrative of the fact that there was no isotopic impurity in the internal standard that could contribute to the 6-oxo - m1dg signal . A representative lc - ms / ms chromatogram of 6-oxo - m1dg and the internal standard [n5]-6-oxo - m1dg isolated from rat feces is displayed . M1dg is an endogenous dna adduct detectable in the genomic dna of humans and rodents . Its presence is associated with peroxidative damage to cellular constituents, particularly polyunsaturated fatty acids, and dna . Although many studies have shown that the lipid peroxidation product, malondialdehyde, can react in vitro with deoxyguanosine to form m1dg, recent studies in e. coli, with defined polyunsaturated fatty acid content in membrane phospholipids, indicate that this is not a major source of m1dg . Rather, the dna peroxidation products, base propenals, appear to account for nearly all of the m1dg generated when bacteria are exposed to peroxynitrite (onoo). Thus, although m1dg appears to be a reliable marker of oxidative damage to cells, it primarily reflects oxidative damage to dna . M1dg is repaired by nucleotide excision repair and is excreted in urine at rather low levels in humans (12 3.8 fmol / kgd). A major contributor to the low levels of m1dg is its oxidative metabolism to 6-oxo - m1dg . This suggests 6-oxo - m1dg may be superior to m1dg as a noninvasive marker of oxidative damage . In the present manuscript, we describe the generation of a monoclonal antibody that exhibits sensitive and specific binding to 6-oxo - m1dg and its use in a quantitative assay suitable for analysis of urine and feces . In the assay, the sepharose - bound mab is used to isolate 6-oxo - m1dg from urine and feces . The purified sample is subjected to lc - ms / ms analysis where 6-oxo - m1dg is quantified via stable isotope dilution against the synthetic internal standard, [n5]-6-oxo - m1dg, which is introduced to the sample prior to antibody purification . The antibody gel shows excellent recovery of 6-oxo - m1dg from buffer solutions . The analyte is recovered at 74% or greater from 10 mm potassium phosphate over a 100-fold range of 6-oxo - m1dg, 0.25 pmol to 25.0 pmol . The internal standard was recovered at 79% (1.0 pmol) from these same solutions, and spiked buffer solutions were quantified accurately to 12% . These data demonstrate that 100 l of the antibody gel is able to bind at least 26 pmol of 6-oxo - m1dg from solution, and lc - ms / ms detection of the purified sample is quantitative . The recovery of [n5]-6-oxo - m1dg from saline solutions was greater than 78% but its recovery from urine and feces was only 29% . This suggests a matrix effect where other nucleosides, deoxynucleosides, and/or endogenous congeners compete with 6-oxo - m1dg for antibody binding sites . Although there was minimal cross - reactivity of the antibody with individual members of a panel of exocyclic adducts and purine oxidation products (figures 1 and 5), it is possible that high levels of low - avidity nucleosides in urine and feces compete with the low levels of endogenous 6-oxo - m1dg for binding sites on the mab . 6-oxo - m1dg was detected in the feces of each of three different sprague dawley rats but was detected in the urine of only one of the rats (table 2). The fecal levels of 6-oxo - m1dg (3501893 fmol / kgday) were significantly higher than the urinary levels (188 fmol / kgday). This is consistent with previous findings that demonstrate m1dg is excreted mainly in urine, whereas 6-oxo - m1dg is excreted mainly in feces . Because 6-oxo - m1dg is present endogenously in rats, it suggests that its metabolic precursor, m1dg, is also produced endogenously in rats and metabolized to 6-oxo - m1dg . This idea is consistent with the finding that m1dg is present in rodent and human genomic dna and with studies from this laboratory demonstrating that 6-oxo - m1dg is the sole metabolite of m1dg . It may be argued that endogenous 6-oxo - m1dg is generated in the nucleoside pool rather than arising from direct damage to dna . However, this seems unlikely if one considers that base propenals are the principal precursors to m1dg . Base propenal formation is triggered by abstraction of the hydrogen from the c-4 position of the deoxyribosyl unit of double - stranded dna . The base propenal produced then reacts with a deoxyguanosine residue in duplex dna to form m1dg . In comparison, the yields of base propenals are much lower when deoxynucleosides are oxidized instead of duplex dna . Furthermore, m1dg formation as a reaction of base propenals and free deoxyguanosine is a bimolecular process . If base propenals are formed in duplex dna, they are generated in close proximity to deoxyguanosine residues in the dna duplex, which should maximize the probability of their reaction with a deoxyguanosine residue . One or both of these compounds may have been ingested, then excreted or metabolized . It was not possible to control for this possibility under the reported experimental regime, so future studies with animals fed nucleic acid - free diets will be required to test this hypothesis . The ability to measure excreted 6-oxo - m1dg has the potential for clinical relevance in assessing the levels of oxidative dna damage in humans . Since the basal levels of both deoxynucleoside adducts are low, they should provide high sensitivity to increases triggered by oxidative damage . Given that the analytical method described herein is applicable to feces and urine, the possibility exists for preclinical or clinical studies conducted in a noninvasive fashion.

A 60-year - old woman (height, 162 cm; weight, 61 kg) visited pain clinic with tactile allodynia and electric shock - like pain in the left dorsal scapular area around the t3 dermatome, which had been diagnosed as phn about 1 month previously and attack of the herpes zoster was 1 year ago . The 100-mm visual analogue scale (vas) of allodynia and electric shock - like pain was rated between 70 and 80 mm on a scale from 0 (no pain) to 100 (worst pain imaginable). The interlaminar epidural block was performed at the t3 - 4 space by the paramedian approach with 5 ml of 0.2% ropivacaine and 20 mg of triamcinolone acetate . Pregabalin and morphine at doses of 150 mg and 10 mg, respectively, twice a day, amitriptyline at a dose of 10 mg before sleep and topical lidocaine patches were prescribed . Dosages of all drugs were adjusted depending on the side effects during the follow - up period . Epidural blocks were repeated twice with a 1-week interval and the continuous intravenous infusion of ketamine (60 mg) was performed over a period of 1 hour twice a week under careful monitoring . After 1 month elapsed, electric shock - like pain was reduced to a vas score of 30/100, but allodynia was not diminished (vas score of 70/100). After 4 months elapsed, we decided to administer magnesium sulfate via the intravenous route . And it was done with continuous intravenous infusion of 1,000 mg of magnesium sulfate in 50 ml of normal saline for 1 hour . Before and after infusion, the serum magnesium levels were checked . After magnesium therapy, she felt very good about her pain and the vas of allodynia was reduced to 40 - 50 . At 1-week follow - up, she was very satisfied with the treatment and reported the reduction of allodynia on the dorsal scapular area of up to 50% (vas 25 - 30/100). However, the serum magnesium level had increased above normal range (2.3 meq / l to 2.9 although it was below the serum level reveals of the adverse effect, we decided to stop intravenous infusion of magnesium sulfate . For more accurate and safe delivery of magnesium to the target site, we applied magnesium using the transforaminal epidural injection technique . With the patient's informed consent, left t3 tfemi identification of the t3 nerve root sheath and epidural space was performed using contrast media (fig . 1). Then, 100 mg of magnesium sulphate and 1 ml of 0.2% ropivacaine (total volume, 2 ml) was carefully injected . Tfemi was repeated twice with a 1-week interval (total of three times) and the degree of pain decreased gradually during the follow - up period . One week after the last procedure, the vas score of allodynia decreased to 15/100 and all medications except pregabalin were discontinued . The vas was 10/100 throughout 1-month follow - up, and pregabalin had also been tapered . To our knowledge, no previous report has described about the magnesium administration by the transforaminal epidural route in patients with neuropathic pain . Here, in our report of phn patient, this treatment resulted in effective pain relief . Previous studies have demonstrated the anti - allodynic effects of nmda receptor antagonists in neuropathic pain disorders . Among the currently available nmda receptor antagonists, ketamine is the most widely used one for the treatment of neuropathic pain . However, ketamine is not always effective and psychomimetic side effects are frequent . Magnesium can antagonize nmda receptor channels by blocking calcium influx in a voltage - gated manner . Intravenous administration of magnesium is efficacious in the management of various conditions associated with neuropathic pain, including phn . Demirkaya and colleagues revealed 1 g i.v . Mg sulfate is effective in the treatment of migraine attacks and collins and colleagues reported that 70 mg / kg magnesium sulphate infusions in 4 hours for 5 days reduced pain in patients with complex regional pain syndrome . Whether intravenous administration of magnesium can achieve a sufficient concentration in the cerebrospinal fluid to block nmda receptors is unclear and studies have reported on the limited efficacy of magnesium when administered via the intravenous route . Furthermore, even if the dose of intravenously administered magnesium is not sufficient to present toxicity, patients are still at risk of magnesium overdose . Neuraxial administration of magnesium is an " off - label " use, and the safety of this technique in human subjects is still undetermined . However, animal studies showed that intrathecally administered magnesium was free of neurotoxicity, and recent studies have demonstrated the safety of magnesium administration via the epidural or intrathecal route in humans . In fact, the exact site of action of epidurally administered magnesium (i.e., spinal or supraspinal) remains unclear . However, comparison with previous reports regarding intravenous magnesium administration suggested that the low dose epidural magnesium used in our patient was unlikely to result in systemic effects . In conclusion, tfemi showed a favourable result in the treatment of intractable allodynia associated with phn . This study was performed in only a single case, and further investigations are required to determine the efficacy of tfemi in the management of allodynia in patients with phn.

Central retinal vein occlusion (crvo) is a common sight - threatening vascular disease with prevalence rates ranging from 0.1%1 to 0.5%2 in the older adult population . Crvo is characterized by occlusion of the central retinal vein and consecutive damming of the venous blood flow . The occlusion may occur at the level of or posterior to the lamina cribrosa.3 despite its prevalence, the pathogenesis of crvo is not completely understood . A combination of vascular, anatomic, and inflammatory factors may contribute to its pathophysiology.4 current treatment options include intravitreal injections of steroids5 or anti - vascular endothelial growth factor (anti - vegf) agents.68 fluorescein angiographic (fa) assessment is the most important tool for an adequate evaluation of the disease severity and for proper classification between the two types of crvo, ischemic and nonischemic . Ischemic crvo is associated with a significant upregulation of vegf, a poor prognosis for visual acuity, and has a higher incidence of secondary neovascular glaucoma (nvg) ranging from 20% to 60% compared to nonischemic crvo with no risk of developing nvg.9 given these significant differences with various outcomes and risk profiles, it is crucial to differentiate between ischemic and nonischemic crvo . Conventional fa images the retinal circulation within the posterior pole . However, it is not known if conventional fa devices are able to capture areas of interest regarding the intraocular metabolism of vegf, such as the peripheral retina . Earlier findings suggested an association between peripheral retinal ischemia and an increased production of vegf.10,11 currently, two ultrawide - field systems are commercially available to perform fa and image the peripheral retina . One method uses a wide - field contact lens system (ocular staurenghi 230 slo retina lens; ocular instruments, inc, bellevue, wa, usa) with the heidelberg retina angiograph (hra; heidelberg engineering gmbh, dossenheim, germany).12,13 the second available system uses the optos scanning laser ophthalmoscope (optos plc, dunfermline, uk), which is a special non - contact - lens - based system that provides visualization up to the peripheral retina.1416 it has been shown that ultrawide - field fa (uwfa) is useful for the assessment of several retinal pathologies17 including retinal vein occlusions,18 diabetes,19,20 uveitis,21 retinal vasculitis,22 choroidal masses,23,24 retinal detachment,25 and retinopathy of prematurity.26 the aim of this study was to evaluate a possible association between peripheral retinal nonperfusion obtained with uwfa and the number of intravitreal ranibizumab injections needed in patients with crvo . This prospective interventional study was performed between june 1, 2012, and february 1, 2014 . The institutional review board of ludwig - maximilians university in munich approved the study design, and patients care adhered to the tenets of the world medical association declaration of helsinki . All patients gave written informed consent for both participation in the study and for fa . Inclusion criteria were diagnosis of crvo (as revealed by retinal hemorrhages and dilated retinal veins in all four quadrants of the fundus) with active center - involving macular edema and central subfield thickness> 250 m detected by spectral - domain optical coherence tomography (sd - oct, heidelberg engineering, heidelberg, germany). Patients without macular edema, with previous focal or panretinal photocoagulation, and with degenerative disorders of the posterior pole and/or retinal periphery were excluded . Additional intravitreal injections were administered in the presence of active center - involving macular edema and central subfield thickness> 250 m, as determined by sd - oct . All patients underwent comprehensive ophthalmologic examination that included best - corrected visual acuity (bcva) measurement, slit - lamp biomicroscopy, applanation tonometry, indirect ophthalmoscopy, and sd - oct before injections and at each follow - up visit . Uwfa using the optos 200t imaging system was obtained in each case before the first injection . All included patients received three intravitreal injections of 0.50 mg ranibizumab (lucentis, genentech, inc ., south san francisco, ca, usa and novartis pharma ag, basel, switzerland) every four weeks and then were examined monthly with follow - up oct scans of the macula . They received additional intravitreal injections if macular edema was detected by sd - oct scans at follow - up visits . Sd - oct volume scans (2015 with 19 horizontal sections, art 9, sd - oct, heidelberg engineering, heidelberg, germany) of the macula were obtained for each study eye to measure the central subfield thickness (cst) in m by using heidelberg sd - oct software; significant macular ischemia was ruled out by uwfa . Fa images were acquired approximately one minute (arteriovenous phase) and 45 minutes (late venous phase) after intravenous injection using the optos 200t scanning laser ophthalmoscope (optos plc) after standard intravenous infusion of 5 ml of sodium fluorescein (10%) by one experienced technician for all included cases . Fa images were compressed into high - quality jpeg files (figures 1 and 2) and analyzed for retinal nonperfusion by two experienced ophthalmologists (kaa and fs). The far peripheral retina was defined as the area between the ampullae of the vortex veins and the ora serrata . The wide - field color images and wide - field fa images were used to identify the vortex vein ampullae, which were located near the ocular equator.27 based on another published work, we defined retinal nonperfusion as at least five disc areas of hypofluorescence28 (representing retinal nonperfusion or capillary dropout) or areas of microvascular pathology (multiple microaneurysms and significant perivascular leakage) on uwfa . Five disc areas was also the mean size of peripheral retinal nonperfusion of all our patients with crvo . Because of the effect of upper and lower eyelid and eyelashes, the far periphery in the horizontal plane alone (the nasal and temporal retina) was analyzed . Collected parameters included demographic information, previous ocular history, number and dates of intravitreal injections, central subfield thickness, peripheral retinal ischemia, central intraretinal fluid, visual acuity, and intraocular pressure (throughout the study period) as well as the occurrence of any complications . Data were collected and analyzed using spss software (version 20.0, ibm corporation, armonk, ny, usa). Spearman rho test was used for correlation analysis . A p - value of <0.05 was considered statistically significant . This prospective interventional study was performed between june 1, 2012, and february 1, 2014 . The institutional review board of ludwig - maximilians university in munich approved the study design, and patients care adhered to the tenets of the world medical association declaration of helsinki . All patients gave written informed consent for both participation in the study and for fa . Inclusion criteria were diagnosis of crvo (as revealed by retinal hemorrhages and dilated retinal veins in all four quadrants of the fundus) with active center - involving macular edema and central subfield thickness> 250 m detected by spectral - domain optical coherence tomography (sd - oct, heidelberg engineering, heidelberg, germany). Patients without macular edema, with previous focal or panretinal photocoagulation, and with degenerative disorders of the posterior pole and/or retinal periphery were excluded . Additional intravitreal injections were administered in the presence of active center - involving macular edema and central subfield thickness> 250 m, as determined by sd - oct . All patients underwent comprehensive ophthalmologic examination that included best - corrected visual acuity (bcva) measurement, slit - lamp biomicroscopy, applanation tonometry, indirect ophthalmoscopy, and sd - oct before injections and at each follow - up visit . Uwfa using the optos 200t imaging system was obtained in each case before the first injection . All included patients received three intravitreal injections of 0.50 mg ranibizumab (lucentis, genentech, inc ., south san francisco, ca, usa and novartis pharma ag, basel, switzerland) every four weeks and then were examined monthly with follow - up oct scans of the macula . They received additional intravitreal injections if macular edema was detected by sd - oct scans at follow - up visits . Sd - oct volume scans (2015 with 19 horizontal sections, art 9, sd - oct, heidelberg engineering, heidelberg, germany) of the macula were obtained for each study eye to measure the central subfield thickness (cst) in m by using heidelberg sd - oct software; significant macular ischemia was ruled out by uwfa . Fa images were acquired approximately one minute (arteriovenous phase) and 45 minutes (late venous phase) after intravenous injection using the optos 200t scanning laser ophthalmoscope (optos plc) after standard intravenous infusion of 5 ml of sodium fluorescein (10%) by one experienced technician for all included cases . Fa images were compressed into high - quality jpeg files (figures 1 and 2) and analyzed for retinal nonperfusion by two experienced ophthalmologists (kaa and fs). The far peripheral retina was defined as the area between the ampullae of the vortex veins and the ora serrata . The wide - field color images and wide - field fa images were used to identify the vortex vein ampullae, which were located near the ocular equator.27 based on another published work, we defined retinal nonperfusion as at least five disc areas of hypofluorescence28 (representing retinal nonperfusion or capillary dropout) or areas of microvascular pathology (multiple microaneurysms and significant perivascular leakage) on uwfa . Five disc areas was also the mean size of peripheral retinal nonperfusion of all our patients with crvo . Because of the effect of upper and lower eyelid and eyelashes, the far periphery in the horizontal plane alone (the nasal and temporal retina) was analyzed . Collected parameters included demographic information, previous ocular history, number and dates of intravitreal injections, central subfield thickness, peripheral retinal ischemia, central intraretinal fluid, visual acuity, and intraocular pressure (throughout the study period) as well as the occurrence of any complications . Data were collected and analyzed using spss software (version 20.0, ibm corporation, armonk, ny, usa). Each graded variable was tested for normal distribution . Spearman rho test was used for correlation analysis . A p - value of <0.05 was considered statistically significant . Fifty - four eyes of fifty - four consecutive patients with treatment - nave crvo were enrolled in this study . Twenty - eight patients (52%) were male, and twenty - three eyes (42.6%) were located on the right side . Seven patients (12.9%) had known glaucoma, 36 patients (66.6%) had systemic hypertension, and 22 patients (40.7%) were pseudophakic . All patients had center - involving macular edema confirmed by macular leakage seen in fa and retinal thickening in sd - oct images . Tables 1 and 2 show the baseline and final bcva and cst . As shown in the tables, there were significant improvements in the visual acuity within and between groups, whereas changes in cst were not significant . Twenty - four eyes (44%) showing less than five disc areas of retinal nonperfusion (group 1) that received a mean number of 4.122.73 intravitreal ranibizumab injections . Thirty eyes (56%) showed more than five disc areas (group 2) of retinal nonperfusion in uwfa and received a mean number of 9.323.84 intravitreal ranibizumab injections (p<0.001, mann whitney u - test). There was a significant positive correlation between the size of peripheral retinal nonperfusion measured in terms of disc areas and the number of injections in each group . There was also a significant correlation between the size of retinal nonperfusion and final bcva in each group . There was no significant correlation between the size of retinal nonperfusion and final cst in each group . We did not observe any eye with neovascularization of the disc or neovascularization elsewhere during the observation period; therefore, panretinal photocoagulation was not performed during this study . Furthermore, no cases of injection - related adverse events like retinal detachment or endophthalmitis were encountered . Venous occlusive disease of the retina is the second most common retinal vascular disorder after diabetic retinopathy.29 it typically affects patients between 40 and 80 years of age.30 usually, a decrease of visual acuity is the result of macular edema, which can lead to permanent visual loss and even legal blindness in severe cases . Applying uwfa, we found a significant correlation between the total number of intravitreal ranibizumab injections and the size of nonperfusion in the peripheral retina: crvo patients with peripheral retinal nonperfusion received more intravitreal injections for treatment of their macular edema than patients without peripheral retinal nonperfusion . We hypothesized that nonperfused peripheral retina could be a source of increased intraocular vegf levels with consecutive macular edema . This observation could explain the requirement for more frequent intravitreal anti - vegf injections of in crvo patients with macular edema and peripheral retinal nonperfusion . This finding is important because an evaluation of the peripheral retina using uwfa may be a prognostic factor and allows the ophthalmologists to estimate the requirement for more or fewer intravitreal injections based on the peripheral retinal perfusion status . Looking at the perfusion of the peripheral retina in those patients, a more precise evaluation of the estimated treatment costs within a defined health care system may be feasible, including a better evaluation of injection - related complications such as endophthalmitis or other complications . Interestingly, the lack of observed neovascularizations in any of the patients even with extended areas of peripheral retinal nonperfusion might be attributed to good patient compliance, strict follow - up visits every 4 weeks, and immediate treatment in case of detected macular edema . The results of our study differ from those published by spaide31 and singer et al.32 spaide evaluated 22 patients treated with ranibizumab for crvo and found that the area of peripheral nonperfusion was not correlated with the number of injections . The follow - up time of his study was longer than ours, but the sample size was smaller . Singer et al32 evaluated 32 patients with retinal vein occlusion and refractory macular edema using sd - oct and uwfa . Those patients were retreated with intravitreal injections with anti - vegf and with dexamethasone intravitreal implant . There was no significant difference in the number of anti - vegf (1.8 vs 1.6, p=0.438) or dexamethasone implant (1.4 vs 1.6, p=0.364) treatments given between the> 10% and 10% nonperfusion groups, nor was there any difference regarding the time to recurrence (3.4 vs 4.3 months, p=0.440). In singer et al s series, there were 13 crvo patients with previously failed contiguous therapy with intravitreal ranibizumab and dexamethasone implant . In contrast, we enrolled previously untreated (treatment - nave) patients; furthermore, the number of our cases was higher than in the abovementioned studies . We previously reported that peripheral retinal nonperfusion correlates significantly with intravitreal ranibizumab injections in patients with brvo and macular edema.33 rehak et al34 evaluated 22 crvo patients in a randomized clinical trial and suggested the selective laser photocoagulation of peripheral areas of nonperfusion may further improve the visual outcome and decrease the number of needed ranibizumab reinjections in crvo patients . There are only few published articles regarding the size of peripheral retinal nonperfusion using uwfa . We calculated the mean size of peripheral retinal nonperfusion for all enrolled patients, which was five disc areas, and then the patients were divided into two groups according to that cut - off point . However, the authors totally agree that this approach is not an established step due to lack of evidence . Moreover, there are several problems in the interpretation of fluorescein angiograms . The grading of the ultrawide - field angiograms is not uniform because there are eyelash artifacts that influence the entire amount of interpretable fundus, and the graders can also change the clarity of the images that may cause intergrader variability . Another drawback of our study design was that we performed uwfa for each patient at only one single time point . Thus, we were not able to demonstrate potential dynamic angiographic changes in nonperfusion areas over the follow - up time . In conclusion, uwfa is a precise tool for the detection and quantification of retinal nonperfusion which correlated with the number of needed intravitreal ranibizumab injections in our patients with crvo . Retinal nonperfusion could be considered as a prognostic factor for a more precise patient management.

Small bowel obstruction (sbo) is one of the most feared complications after a gastric bypass . It can be chronic or acute and can range from a nuisance to the patient to a life - threatening emergency sbos related to adhesions occur after laparoscopic roux - en - y gastric bypass (lgb), but ih formation, secondary to the reconstruction of the small intestine, is also of concern to bariatric surgeons . Multiple defects in the mesentery of the bowel can occur and may lead to ih and, in an antecolic bypass, a defect is associated with the roux limb passing over the transverse colon . The reported incidence of sbo after lgb varies widely, perhaps because the technique used in the operation also varies widely, not only in placement of the roux limb, but regarding closure or nonclosure of the mesenteric defects . The disparity in reports leads to confusion regarding the true incidence and even the causes of sbo after lgb . In addition, the closure of mesenteric defects is questioned in a substantial body of literature . The objective of this study was to examine the incidence and characteristics of sbo after antecolic antegastric bypass with nonclosure of the mesenteric defect of the jejunojejunal (jj) anastomosis . Following health insurance portability and accountability act guidelines, the author performed a retrospective chart review of a series of consecutive lgbs performed over a 3-year period and recorded in a prospectively maintained database . The follow up of the patients included office visits at 1 week, 1 month, 3 months, 6 months, and then, yearly . All procedures were performed by the same surgeon using the antecolic antegastric technique . In no case was the mesenteric defect closed . Any patients presenting with signs and symptoms of an sbo were emergently taken to the operating room . The policy of our bariatric program has always been to explore aggressively any patient with suspicion of an sbo, whether based on physical examination, radiographic studies, or history . Patients with an sbo were usually taken to the operating room from the emergency department . Those who underwent revisional bariatric surgery or conversion to open operation during the primary surgery were excluded . Revisional procedures were not included because adhesions may have formed from the previous bariatric surgery that could have confounded the results . All patients had given informed consent prior to surgery for their deidentified data to be used for study purposes . The gastric pouch was sized to 20 ml with an orogastric balloon with 4 to 5 staple loads with seamguard (w. l. gore, flagstaff, arizona) staple - line reinforcement (slr) and the echelon 60-mm stapler (ethicon endo - surgery, cincinnati, ohio). The small bowel was divided 40 cm from the ligament of treitz, and the mesentery had minimal division . The only division of the mesentery was from the same staple load that divided the small bowel . The roux limb was measured to 100 to 120 cm, depending on the body mass index (bmi) of the patient . The gastrojejunal anastomosis was hand sewn in 2 layers with absorbable sutures over a 34 french bougie . The jejunojejunostomy was formed with a single firing of the same stapler, and the enteroenterostomy was closed with the stapler . In all cases, an intraoperative endoscopy was used to check the gastrojejunal anastomosis, and drains were used in all male patients because of higher technical difficulty and higher rates of mortality and morbidity; in patients with a bmi over 50, for the same reasons; and in selected cases . There was one conversion to an open operation for an incomplete malrotation; there was no mortality . The gastric pouch was sized to 20 ml with an orogastric balloon with 4 to 5 staple loads with seamguard (w. l. gore, flagstaff, arizona) staple - line reinforcement (slr) and the echelon 60-mm stapler (ethicon endo - surgery, cincinnati, ohio). The small bowel was divided 40 cm from the ligament of treitz, and the mesentery had minimal division . The only division of the mesentery was from the same staple load that divided the small bowel . The roux limb was measured to 100 to 120 cm, depending on the body mass index (bmi) of the patient . The gastrojejunal anastomosis was hand sewn in 2 layers with absorbable sutures over a 34 french bougie . The jejunojejunostomy was formed with a single firing of the same stapler, and the enteroenterostomy was closed with the stapler . In all cases, an intraoperative endoscopy was used to check the gastrojejunal anastomosis, and drains were used in all male patients because of higher technical difficulty and higher rates of mortality and morbidity; in patients with a bmi over 50, for the same reasons; and in selected cases . There was one conversion to an open operation for an incomplete malrotation; there was no mortality . There were 249 primary lgbs performed in the study period (january 1, 2011, through december 31, 2013). Sbo in 4 cases was caused by ih (incidence of 1.6%) and by adhesions in 11 (73%) (figure 1). In the cases with ih, the most common locations were from the jj to the roux limb (n = 5), followed by those forming from the jj to the abdominal wall (n = 3), and 1 each from the jj to the colon and from the jj to the common channel (figure 2). In all cases, the adhesions were treated with laparoscopic lysis . The average time to sbo was 8.1 months (range, 121) from the initial surgery . Sbo is common after lgb and can be easily managed or can just as easily lead to disaster if not recognized and treated promptly . The surgical literature has many articles that discuss ih formation, and an area of contention has been closure versus nonclosure of the mesenteric defects . Most authors seem to agree that if a retrocolic gastric bypass is performed, the mesocolic defect must be closed, which can lead to a 0% rate of ih formation, although that number can climb as high as 15% . Elms et al showed that, in almost 2 400 patients who underwent antecolic antegastric bypass, ih formed in only 1.1%, primarily at the mesenteric defect . In those cases the defects had all been closed . Cho et al had a 0.2% ih formation rate in 1 400 patients who underwent antecolic antegastric bypasses with no mesenteric closure and no division of the mesentery . Rodriguez et al also reported that minimal division of the mesentery could lead to a decrease in ih formation . They showed that, with closure of the defect but with wide opening of the mesentery, there was a 14.4% ih rate; if the defect was closed and the mesentery was not widely opened, the ih rate dropped to 1.1% . Abasbassi et al showed a lower ih formation rate with no division of the mesentery, but also no closure of the mesentery . This cumulative experience could indicate that a way to decrease ih formation is to perform a minimal division of the mesentery . Obeid et al used mixed techniques in both retro- and antecolic bypasses, some with closure of defects, some without . In 679 patients with an antecolic roux limb, the majority of all the defects closed . In the retrocolic group, almost 4% of these had closure of the mesenteric defect, 8.4% had nonclosure; 3.8% were in antecolic and 8.5% in retrocolic bypasses . This wide range of techniques makes an overall analysis of the literature difficult . In 2010 in jsls, hope and colleagues examined the incidence of ih formation after documented closure of mesenteric defects . Permanent sutures were used to close these defects, but 15 of 18 patients in the series presented with open defects, causing ih . These outcomes indicate that closure of the defects may not be as permanent as surgeons would like to believe, and indeed this very situation was the catalyst for this paper . In our lgbs, we were closing all defects, but our ih formation rate seemed to be climbing . We noticed most of the mesenteric defects were open at reoperation, and so we switched to nonclosure . As demonstrated here, the published papers on the subject have reported a wide range of ih incidence, from 0% to 15.5% . Another complicating factor may be that many of the papers published in this area are from surgical programs training fellows, and the association of postsurgical complications with performance of operations by fellows is not known . In this study, the author specifically examined the incidence and cause of sbo in antecolic antegastric bypasses . In no case was either the mesenteric or antecolic defect closed . The interesting finding was that, of an overall incidence of sbo of 6%, only 1.6% of the total was caused by ih formation, all at the jj mesenteric defect . This outcome compares favorably with the ih formation rate reported by other authors who did or did not close the defects . Also, the incidence of sbo caused by adhesions is higher in the current study than in some others . Elms et al reported that 47.6% of the sbos in their series were secondary to adhesions, mostly at the jj, compared with our rate of 73% . The opposing end of the adhesion had multiple locations, but the staple line of the jj was always at one end, perhaps because of the intense inflammatory reaction that it generates . A hand - sewn technique could be used to close the common enterotomy and might reduce the incidence of sbo slightly, but the cut end of the biliopancreatic limb still has an exposed staple line . The strength of this study is in the homogeneity of the technique used, as this was a single - surgeon series . However, its strength is also its chief weakness, as the results may not be reproducible by others . The number of sbos is also small enough to raise the possibility of a type ii error . The author lives in a geographically isolated part of the country, with no other surgeons who are willing to care for bariatric patients, and so it was more likely that all the sbos would be treated by the same bariatric surgeon . Another limitation of the study is that it was a small series with a short follow - up . Although only the years of 2011 through 2013 were examined, and the average time to sbo was 8.1 months, it is possible that the sbo rate will climb in these same patients as time passes, and some of the patients are likely to be lost to follow - up . Another confounding factor is that an slr was used early in the series at the jj, but not in later operations . There may be an association of slr with adhesion formation, but the connection is not clear . The most common location of adhesions was at the jj, were slr was used, but about half of the sbos caused solely by adhesions to the jj occurred after the use of slr was abandoned, so there are probably not enough cases to tell if the use of slr increases sbo . The usual weaknesses of a retrospective study apply to this paper as they do to all other studies about ih formation . Surgeons should remember that the mainstay of treatment of sbo after lgb is an operation . An isolated roux limb obstruction can be decompressed by vomiting, and the placement of an ngt involves risk of a perforation . The surgeon should move the patient quickly to the operating room once an sbo is diagnosed or even suspected, as bowel ischemia can lead to extensive bowel resection and nutritional debility or even death . These operations can often be performed laparoscopically, but laparotomy is also safe and effective . The incidence of sbo caused by ih with nonclosure of the mesenteric defect is similar to that in other series where the defect was closed . Regardless of the cause of the sbo, operation remains the definitive treatment and should not be delayed in the gastric bypass patient.

The following section describes the treatment procedure for patients with deep infection following tka using modified static spacers . The original prosthesis was removed, followed by intensive irrigation and wide debridement of the infected soft tissue . A 36 fr - diameter straight thoracic catheter (mallinckrodt medical, athlone, ireland) and a steinmann pin measuring 3.0 mm in diameter and 22 cm in length vancomycin (2 g) was then added to the gentamicin bone cement (depuy, warsaw, in, usa). At the late liquid stage of the cement, the steinmann pin was inserted into the tube, and the prepared cement was delivered through the tube . During this procedure, the tube was removed from the cement rod using a surgical knife . Finally, a cement rod measuring 9 mm in diameter and 22 cm in length was formed (fig . An entry hole was created at the center of the distal femur and proximal tibia for the insertion of the cement rod . The rod was then inserted into the femur and tibia through this hole . During insertion, it was important to place the center of the cement rod on an imaginary joint line . The proximal medullary canal of the tibia was filled with antibiotic - impregnated cement up to the surface of the proximal tibia, while the surgical assistant maintained proper anatomic alignment and joint space . The space between the cement of the distal femur and the proximal tibia was filled with more antibiotic - impregnated cement . Finally, the suprapatellar pouch and medial and lateral gutter space were filled with antibiotic - impregnated cement to reduce soft tissue adhesion (fig . Cylinder splint immobilization was required for three days after the operation, after which a fixed - angle knee brace was used and toe - touching ambulation was allowed until the reimplantation surgery . From april to november 2007, the authors performed the static technique in four patients using the novel antibiotic - impregnated cement rod for the treatment of infected tka (culture: staphylococcus in three cases and no bacteria in one case). Follow - up laboratory studies, including erythrocyte sedimentation rate and c - reactive protein, culture study via knee aspiration and a frozen biopsy from the second - stage operation (<5 polymorpho - leukocytes / high power field) were performed to confirm successful eradication of the infection . The second - stage reimplantation was performed when all the criteria for the validation of infection control were met (fig . The second - stage reimplantation was performed using the rectus snipping approach, and more than 90 of flexion was obtained intra - operatively . In all four patients, no re - infection was evident after two and a half years of follow - up . The range of motion of the knee joints were respectively improved from 50 to 80, 95, and 100, and the knee society scores were 70, 86, 65, and 84, respectively, in the last follow - up evaluation . Fehring et al.8) emphasized the importance of resting the joint in septic joint conditions . Others also reported that the static spacer technique provides more stability than the mobile spacer technique in patients with severe bone loss.5,8) the main advantage of this technique is the maintenance of a normally aligned lower limb during the interval period . This maintains knee stability in combination with early muscle strengthening exercises, including quadriceps setting exercise, and enables the patient to comfortably dress and manage him / herself during the intervening period . The cement rod and static spacer provide a stable gap between the femur and tibia, thereby minimizing soft tissue contracture and shortening of the lower limb . In addition, symmetric maintenance of the soft tissue of both medial and lateral gutters requires no additional soft tissue balancing at the second - stage reimplantation . The additional cost of a metal nail, however, and the difficulty in infection control due to biofilm formation around the metal nail can be troublesome . In addition, a metal nail cannot be removed easily at the second stage reimplantation due to its hardness, and soft tissue adhesion of the femoral or tibial medullary canals . On the other hand, an antibiotic - impregnated cement rod enjoys advantages, such as inexpensive antibiotic delivery to the marrow spaces and easy removal by using a hercules cutter . In addition, a static spacer is anchored to the cement rod, which can prevent spacer migration and bone erosion . This feature is believed to generate less cement wear debris than with the conventional static spacer or mobile articulating spacer technique . During the conduct of the antibiotic - impregnated cement rod technique, antibiotic - impregnated cement was applied to the proximal tibia, distal femur, joint gap space, suprapatellar pouch, and both gutters in a stepwise manner . This technique enables easy removal of the cement and reduced soft tissue adhesion at second - stage reimplantation.

Mental fatigue is common and can be a disabling long - time condition following a stroke . It has been estimated that about 3070% of stroke survivors complain of fatigue [17]. Even for those with an almost recovered stroke and without neurological and neuropsychological impairments, the person who suffers from mental fatigue is able to perform mental effort just for short periods, and, notably, it will take longer than normal to regain energy after being exhausted . Accompanying symptoms, such as irritability, sensitivity to stress, concentration difficulties, and emotional instability may further impair social interactions [811]. However, attention has been paid to poststroke fatigue during the last 10 years, and fatigue is now generally held as a separate phenomenon [46, 1214]. Few investigations have been carried out to evaluate fatigue and cognitive functions following a stroke . Leegard reported that fatigue is frequent following a stroke but did not find any related impaired cognitive functions . Van zandvoort and coauthors investigated lacunar infarct and reported frequent difficulties relating to fatigue and a decreased cognitive performance under more demanding conditions . With the intention to increase knowledge about mental fatigue and cognitive difficulties related to stroke, we examined well - rehabilitated stroke participants, who had suffered from long - term mental fatigue for at least one year prior to examination . The subjects were examined for subjective self - reporting of mental fatigue, depression, and anxiety symptoms . Neuropsychological tests were aimed at evaluating information processing speed, attention, and working memory . Twenty - four participants, having recovered from neurological symptoms, but suffering from pathological mental fatigue for at least one year following a stroke and 24 healthy controls were included in the study . The study persons were recruited from an advertisement in a local, daily newspaper or from the neurological clinic at the local university hospital and were later included in intervention studies . The stroke subjects should have been healthy and at work before the stroke meaning that they had no known diseases but hypertonia was present among some participants . The type of stroke was obtained from medical records and self - reports (see table 1). The control participants were recruited from the local community, with no history of brain injury, stroke, psychiatric or neurological disorder, and no drug abuse, and they were fully able to work . They performed cognitive tests focused on information processing speed, attention, and working memory . The self - assessment for mental fatigue is a multidimensional questionnaire containing 15 questions and is adapted from rdholm et al . . The self - reported questionnaire covers the most common symptoms occurring after brain injury, stroke, or other neurological disorders affecting the brain [11, 17]. The self - assessment scale for mental fatigue and related items has been evaluated, and the 14 questions had adequate internal consistency with a cronbach's alpha of 0.94 . The questions concern fatigue in general, lack of initiative, mental fatigue, mental recovery, concentration difficulties, memory problems, slowness of thinking, sensitivity to stress, increased tendency to become emotional, irritability, sensitivity to light and noise, and decreased or increased duration of sleep as well as 24-hour variations . This cprs scale is used here for self - assessment of depression and anxiety . The neuropsychological tests included digit symbol - coding from the wais - iii, measuring information processing speed, digit span from the wais - iii, measuring attention and working memory; verbal fluency test, fas; trail making test (tmt) a and b, measuring visual scanning, divided attention and motor speed . In order to evaluate higher demands such as dual tasks, a series of two new trail making tests was constructed with three and four factors, respectively . Months were added in part c and both months and days of the week in chronological order in part d. in the latter, the order of letters and digits was switched . A new computer test was constructed in our department including a single and a complex subtest . The single test included speed of mouse click in four squares, located in each corner of a bigger square (6 6 cm) on the computer screen and was performed in a clockwise order . A mouse click outside the square was recorded as a miss, and a new click was necessary to do in order to be able to go on with the test . The complex sub - test also included the same mouse clicking procedure, but at the same time, the subject was asked to count how many instances of a specific digit between zero and nine, randomly chosen, he / she could see . After the 30 seconds, the subject was asked to report how many of the specific digit he / she had seen . It was possible to measure the difference in speed between a single and a complex task, variability over time, and errors made in counting digits . In the complex test, it was necessary to attend to both the square and the digits . A comparison between groups was done by t - test and analysis of covariance (ancova). The mann - whitney u test was used when analyzing separate items included in the self - assessment scales . Pearson's correlation and linear regression were used for analysis of connections between variables . Spss 16.0 for windows the control group had significantly more years of education than the stroke group (t - test, p = 0.001), and age was almost significantly different when comparing the two groups (p = 0.055, table 1). Accordingly, ancova, controlling for the variance for education and age, was conducted for all the variables analysed . The only significant gender difference found was that the control females were faster in the digit symbol - coding test, and an ancova, also controlling for sex, was conducted for this variable . A significant difference was found between the groups for the total sum of scores for mental fatigue . The mean value for the stroke group was 18.4 with a 95% confidence interval of between 16.420.5 . The mean value for the control group was 4.0, with a 95% confidence interval of between 2.95.0 (see figure 1). The total score for the cprs scale, taken from the depression and anxiety subscale, was also rated significantly higher in the stroke group compared to controls (see table 2, figure 1). The significant effect for mental fatigue remained after adjustment for depression (p <0.001). All the separate items in the self - assessment scale for mental fatigue were rated significantly higher for the stroke group compared to controls . The cprs gave the following findings: without taking into account overlapping items, the items relating to sadness, emotional involvement, pessimistic thoughts, and zest for life rated significantly higher for the stroke group (adjusted for multiple comparisons, as shown in figure 2). Among the stroke subjects, 74% reported a clear 24-hour variation with morning most frequently reported to be the best time of the day and afternoon and evening the worst . The participants from the stroke group were significantly slower on the test measuring information processing speed, primarily digit symbol - coding, and also reading speed and number of mouse clicks in the computer test . The stroke group was also significantly slower and made more errors in tmt d, the most demanding of the tmt tests . They were also significantly slower on tmt b, and produced fewer words on the verbal fluency test . The result for the simple mouse click sub - test was found to be significantly faster for the controls compared with the stroke subjects . The computer test which placed a simultaneous demand on speed, attention, and working memory, resulted in a fairly good speed for the stroke subjects, with no detected difference in speed compared to the controls . On the contrary, the stroke subjects made significantly more errors compared to the controls (table 2, figure 3). The cognitive tests with significant results (p <0.05, see table 2) were included in a linear regression model, using the enter method . Digit symbol - coding (p = 0.004) and the scores for number of errors in the computer test (p = 0.018) were significant predictors for mental fatigue, while the other scores were not significant predictors for mental fatigue . Digit symbol - coding and number of errors in the computer test also correlated significantly to the mental fatigue sum of scores (r = 0.59 and r = 0.46). The total scores for cprs, depression, and anxiety were on a significantly higher level compared to controls . The madrs, which provides a separate scale, has the same format as the cprs self - assessment scale depression, except that the items are graded in a different way . In the cprs scale, depression, the highest level is three for each separate cprs item, while the levels for the alternatives in the madrs scale are double for each separate item, with the highest level being 6 . According to madrs, a score between 12 and 20 is regarded as mild depression and 21 and above indicates a probable true depression . The mean level of eight for the stroke subjects corresponds to 16 on madrs, indicating an overall mild level of depression . There was a highly significant difference between the groups on their self - assessment of mental fatigue, with a mean value of 18 reported for the almost recovered stroke group and a mean value of four for the control group . The self - assessment scale has no cut - off value for fatigue, but our experience shows that a value of 15 and above indicates a clear problem with mental fatigue [9, 23]. The clear difference in 24-hour variation also showed the specific exhaustion mental fatigue subjects experience during the active time of the day . It should be noticed that stroke victims were included due to the presence of mental fatigue during one year or more following the stroke . The data we have presented in our study do not include an indication of the frequency of mental fatigue after different types of stroke . However, on an individual basis, according to madrs, eight participants were not depressed, seven had an indicated mild depression, and nine had a probable, true depression . Among the controls, two participants scored on a mild depression level, while the remainder scored below this level . The total sum of scores on the depression scales may be deceptive if a person is complaining of concentration difficulties and fatigue, but does not have a depressed mood, nor a lack of interest in and enjoyment of daily activities . In this study, three items were overlapping between mental fatigue and depression, and these items were rated on a higher level than the corresponding specific items on the depression sub - scale (see figure 2). Depression and mental fatigue can occur on their own, but they sometimes occur simultaneously, as shown in this study . Accordingly, we suggest mental fatigue and depression to be independent phenomena following a stroke . This also conforms with the findings from other studies [46, 12, 13]. The distressing exhaustion along with the bad memory, concentration difficulties, and not being able to perform simultaneous tasks are the phenomena many subjectively complain of following a stroke . It is important to include cognitive tests in the examinations in order to better understand the difficulties connected to mental fatigue and for the purpose of recommending treatment strategies . In this study, physically well - recovered stroke subjects, who had no medical problems except long - term mental fatigue, also showed decreased information processing speed and made more errors in demanding cognitive tasks compared to the control subjects . Processing speed is also fundamental and important when considering cognitive functions of a higher order . Few studies have been carried out which cover fatigue and cognitive performance following a stroke . However, studies have shown the connection between fatigue and decreased cognitive performance under more demanding conditions, while no cognitive impairments were detected, despite frequent fatigue . With more demanding and sensitive tests, including processing speed and complex attention, it may be possible to detect cognitive impairments accompanying mental fatigue . This is important, as mental fatigue and cognitive deficits will be an obstacle to almost recovered stroke subjects who are on their way to a return to work and previous activities, as every - day life today is complex, with high demands being placed on simultaneous and rapid decisions . In conclusion, mental fatigue is disabling for many people following an almost recovered stroke, and this is suggested to be related to cognitive impairments, primarily information processing speed, and attention . Mental fatigue should also be treated as a separate phenomenon and should be differentiated from, and not confused with, depression . Today, no specific guidelines exist for the treatment of mental fatigue, and research is urgently needed for this common yet distressing symptom.

Cigarette smoke (cs) is associated with the development of inflammation - related diseases such as chronic obstructive pulmonary disease and vascular diseases, including atherosclerosis and stroke . Several studies have revealed that cs is a major contributor to vascular diseases because it accelerates the development of atherosclerotic plaques . The relationship between cs and the increased incidence of atherosclerosis has been reported [57], which may be a consequence of direct endothelial damage, increased proliferation of smooth muscle in atherosclerotic lesions, and/or decreased vasodilation . Endothelial damage has also been suggested as the initial cause of development of vascular diseases . In a previous study, it was shown that inhibition of oxidative stress exerts protection in human endothelial cells, which could be an effective strategy in the treatment of vascular diseases . A number of studies support that reactive oxygen species (ros) causing oxidative stress may play an essential role in mediating endothelial cell death . Oxidative stress is a major factor in vascular diseases such as hypertension, stroke, and atherosclerosis . Korean red ginseng (krg) is a popular traditional herbal medicine that has been widely used to treat several diseases such as cancer and vascular diseases . Recent research shows that ginseng may have therapeutic potential in the treatment of alzheimer's disease, diabetes, cancer, and cardiovascular diseases, through its antioxidant, antithrombotic, antihyperlipidemic, and anticancer effects [1215]. In endothelial cells, krg simulates no production in vivo and in vitro, suggesting that krg has antihypertensive effects . Krg also promotes angiogenesis through the activation of the signaling pathway, indicating that krg can be implicated in potential angiogenic therapies for improving tissue repair, wound healing, and cardiovascular diseases . In addition, our previous study suggested that krg exerts a cytoprotective effect through the induction of heme oxygenase (ho)-1 expression, suggesting a possible therapeutic mechanism of krg in cardiovascular diseases . It is well known that chronic inflammation contributes to the pathogenesis of many human diseases such as atherosclerosis . Accumulating evidence suggests that krg is involved in the regulation of inflammatory responses, suggesting an anti - inflammatory effect of krg . Cyclooxygenase (cox) catalyzes the conversion of arachidonic acid to prostaglandins that play vital roles in multiple physiological and pathophysiological processes, including inflammation . In particular, cox-2 is normally undetectable in most tissues and is induced in response to numerous stimuli . Vascular diseases may, in part, be caused by cox-2 upregulation at sites of inflammation and vascular injury . Cox-2 plays an important role in inflammation, therefore, inhibition of cox-2 expression may participate in the treatment of inflammation - related diseases such as vascular diseases . The objective of our study was to investigate the vascular protective effect of krg in acrolein - stimulated human umbilical vein endothelial cells (huvecs). Therefore, we examined the involvement of cox-2 expression via p38 mitogen - activated protein kinase (mapk), intracellular ros, and apoptosis in acrolein - stimulated huvecs . Krg powder was obtained from the korea ginseng corporation (daejeon, korea). M199 medium and fetal bovine serum krg powder was soaked in water (1:25, w / w) for 3 h, and boiled for 40 min . Following centrifugation at 1,900 g for 60 min, supernatants of ginseng extract were further centrifuged at 10,000 g for 30 min and lyophilized . The general composition of the product offered by the korea ginseng corporation is as follows: moisture 36%, solid volume 64%, ash 2.5%, total fat 0.05%, total crude saponin 70 mg / g, and total ginsenosides 20 mg / g . Huvecs were maintained in m199 medium and supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin, 10 ng / ml human fibroblast growth factor, and 18 mu / ml heparin . Huvecs were grown to 80% confluence, maintained with fresh medium described above, and subcultured every 2 or 3 d. the cells were used within nine passages during these experiments . We applied 20 or 40 g of the whole cell lysate proteins to each lane and analyzed them with western blotting . Western blotting was performed using primary antibodies as follows: anti - cox-2, p38 mapk, phopho - p38, cyclic amp - responsive element - binding protein (creb), phospho - creb (cell signaling, danvers, ma, usa) and anti - glyceraldehyde 3-phosphate dehydrogenase (abfrontier, seoul, korea). Horseradish - peroxidase - conjugated anti - igg antibodies were used as the secondary antibody to detect the above - mentioned protein bands by enhanced chemiluminescence westsave - up (abfrontier). The rna pellets were washed in 70% ethanol, dried completely, and dissolved in diethylpyrocarbonate to inhibit rnase . Total rna was quantified using a nd-100 spectrometer (nanodrop technologies, wilmington, de, usa). Polymerase chain reaction (pcr) was performed using the synthesized cdna as a template and using specific primers for cox-2 or -actin as a loading control . The primer sequence for human cox-2 was 5-gacagtccaccaacttacaat-3 (forward) and 5-catctctccatcaattatctgat-3 (reverse). The amplified products were resolved by 1% agarose gel electrophoresis, stained with ethidium bromide, and photographed under ultraviolet light . Huvecs were cultured in a glass culture chamber slide (falcon plastics, london ontario, canada) and processed for immunofluorescence analysis . The amount of prostaglandin (pg)e2 in the culture medium was measured using the pge2 eia kit according to the manufacturer's protocol (cayman chemical company, ann arbor, mi, usa). Samples as well as standards were applied to a 96-well plate, precoated with goat anti - mouse igg, and incubated with pge2 acetylcholinesterase tracer and pge2 antiserum . All the wells were emptied, rinsed five times, and incubated with ellman's reagent for 60 min in the dark with gentle rocking to produce 5-thio-2-nitrobenzoic acid, which has a strong absorbance at 405 nm; the plate was read at 405 nm in an enzyme - linked immunosorbent assay reader (el 800; bio - tek, winooski, vt, usa). We calculated the results using the standard curve, which were expressed as picograms per milliliter . Intracellular ros in acrolein - stimulated huvecs is analyzed using a fluorescent dye, 2,7-dichlorofluorescein diacetate (dcf / da). In the presence of oxidants, after 18 h incubation with 25 m acrolein in the presence or absence of krg, cells were stained with 10 m dcf / da, and fluorescence was analyzed by a facs vantage flow cytometer (becton dickinson, san jose, ca, usa) and fluorescence microscopy (eclipse 50i; nikon, japan). To clarify whether krg - mediated inhibition of acrolein - induced cox-2 expression plays a significant role in cytoprotection against oxidative stress, acrolein - stimulated cells were pretreated with krg (1 mg / ml) or untreated, and cell death was measured by in situ terminal transferase dutp nick end labeling (tunel) assay . To measure dna fragmentation, the commercially available in situ death detection kit (roche diagnostics, mannheim, germany) huvecs were cultured in a glass culture chamber slide and fixed for 30 min in 10% neutral buffered formalin solution at room temperature . A tunel assay system was used, according to the manufacturer's instructions, for examination under a fluorescence microscope, with excitation at 488 nm and emission at 525 nm . Fluorescein isothiocyanate (fitc) (bd pharmingen, san diego, ca, usa) and propidium iodide (pi) staining of necrotic and apoptotic cells . Cells were washed in pbs, resuspended in 100 l binding buffer containing 5 l annexin v fitc and 1 g / ml pi, and incubated for 10 min at room temperature in the dark . Positioning of quadrants on the annexin v / pi dot plots was performed as previously described . Data were expressed as mean standard deviation . Statistical analysis was performed using one - way analysis of variance (graphpad prism version 4; graphpad software, san diego, ca, usa) followed by bonferroni's multiple comparison test . A previous study found that acrolein in cs induces cox-2 expression in human endothelial cells . Krg inhibited acrolein - induced cox-2 protein expression in a concentration - dependent manner (fig . After pretreatment of acrolein - stimulated cells with krg, the cells were fixed, and cox-2 localization in huvecs was observed by immunofluorescence staining with an anti - cox-2 antibody followed by a fluorescence - tagged secondary antibody . Immunofluorescence analysis showed that acrolein - induced cox-2 protein levels were inhibited in huvecs after treatment with krg (fig . The induction of cox-2 expression is known to be responsible for pge2 release in the culture medium of cells stimulated with acrolein . Acrolein increased pge2 secretion, which was dramatically reduced by krg (fig . 2). This result indicates that krg leads to the reduction of cox-2 protein expression and subsequently pge2 biosynthesis in acrolein - stimulated huvecs . Thus, we examined the effect of krg on ros production in acrolein - stimulated cells . The shift to the right of the curve due to increased fluorescence indicates an increase in the intracellular levels of ros . The results indicate that ros generation in cells treated with acrolein increased compared to untreated cells, whereas krg inhibited acrolein - induced ros generation (fig . These results indicate that krg may play a role in the inhibition of cox-2 expression via reduction of acrolein - generated ros in acrolein - stimulated huvecs . Thus, to determine the upstream signaling pathway involved in krg - mediated cox-2 inhibition, we measured the activation of p38 and creb by detecting increased phospho - p38 and phospho - creb levels in acrolein - stimulated cells and found that phosphorylation of p38 and creb was strongly reduced by krg in acrolein - stimulated cells (fig . 4). These results demonstrate the role of p38 and creb signaling in the inhibition of acrolein - mediated cox-2 induction . Fluorescence - activated cell sorting showed that while the number of apoptotic cells increased following treatment with acrolein, pretreatment with krg reduced the number of apoptotic cells (fig . To confirm this result, we evaluated the presence of dead cells by tunel staining, which is widely used in detecting dna fragmentations in situ . The tunel assay indicates cell death, including apoptosis, by detection of the appearance of intensely stained nuclei, which indicates incorporation of labeled dutp into the 3-end of fragmented dna derived from apoptotic nuclei . As illustrated in fig . 5b, acrolein treatment significantly increased the proportion of tunel - positive cells, which was restored by krg pretreatment . These results revealed that the vascular protective effect of krg is mediated by the inhibition of cox-2 expression in acrolein - stimulated huvecs . In this study, we explored the inhibition of an inflammatory mediator, cox-2, by krg water extract in huvecs . We found that krg inhibited both mrna and the protein level of cox-2 and its cytoprotective effect in acrolein - stimulated huvecs . There is increasing evidence that,-unsaturated aldehydes in cs, including acrolein and crotonaldehyde play an important pathophysiological role in vascular diseases such as atherosclerosis and alzheimer's disease . Exposure to,-unsaturated aldehydes is critical to the inflammatory response via activation of the proinflammatory signaling pathway and redox - sensitive transcription factors . Furthermore,,-unsaturated aldehydes increase oxidative stress, which plays a crucial role in the pathogenesis of vascular diseases via direct injury to the endothelium . Cox-2, a key enzyme for prostaglandin biosynthesis, is an inducible enzyme that is rapidly induced during inflammatory reactions . Numerous studies have reported the involvement of cs in vascular diseases through cox-2 and endothelial no synthase activity . Chronic inflammation plays an important role in vascular diseases, therefore, cox-2 may participate in the development of inflammation - related diseases, including vascular diseases . Ginseng has been used as a general tonic for> 2000 years in east asia, and it has become a famous herbal medicine for treatment of various diseases, including vascular disorders . Krg has been reported to have effective pharmacological activities, including antioxidant, anticarcinogenic, and ameliorative effects on blood circulation . Recently, the diverse effects of several constituents of krg, including ginsenoside, on endothelial cells have been extensively studied . Hien et al demonstrated the anti - inflammatory and antiatherosclerotic activities of ginsenoside rg3 in human endothelial cells, with a decrease of cell adhesion molecules and proinflammatory cytokines . Moreover, the cytoprotective effect of ginsenoside rb1 in endothelial cell damage mediated by oxidized low - density lipoprotein has been reported . Several constituents of red ginseng have been reported to regulate proliferation and migration and to protect oxidative stress - mediated damage in human endothelial cells . There is evidence demonstrating the presence of major ginsenosides including rb1 and rg1 in krg water extract . Thus, these components could also contribute to the diverse retinue of protective actions of krg . A previous study showed that the induction of ho-1 expression may exert protective effects in krg - treated human endothelial cells . However, there have been no reports revealing the mechanism underlying krg - inhibited cox-2 expression in acrolein,,-unsaturated aldehydes in cs, stimulated huvecs . We have established that the major signaling pathway of cox-2 (i.e., p38 mapk creb) and intracellular ros generation are involved in this inhibition of cox-2 expression in acrolein - stimulated huvecs by krg . As mentioned above therefore, the inhibition of cox-2 expression following krg water extract treatment may be associated with its strong protective effect in acrolein - stimulated huvecs . In conclusion, we propose that the krg water extract may exert a cytoprotective effect through the inhibition of cox-2 induction and that this reduction of cox-2 in acrolein - stimulated huvecs is mediated by the p38 mapk

4cl4-coumarate: coa ligasecas9crispr - associated protein 9crisprclustered regularly interspaced short palindromic repeatsctcondensed tanningrnaguide rnammillionpamprotospacer adjacent motifpol iiirna polymerase iiirnairna interferencernaseribonucleasesnpsingle nucleotide polymorphisms / g ligninsyringyl - to - guaiacyl lignin ratiotrnatransfer rna 4-coumarate: coa ligase crispr - associated protein 9 clustered regularly interspaced short palindromic repeats protospacer adjacent motif single nucleotide polymorphism syringyl - to - guaiacyl lignin ratio since the first report of its programmable gene editing potential, the crispr / cas9 technology is revolutionizing all facets of biology from medicine to agriculture (jinek et al ., 2012). With its efficiency and simplicity, crispr / cas9 has quickly displaced its predecessors (e.g., zinc finger and transcription activator - like effector nucleases) as the method of choice for genome editing (carroll, 2014). For agricultural applications that have traditionally depended on gene - silencing for rna modification, crispr / cas9 is a game changer . The previous methods, such as antisense or rna interference (rnai), leave much to be desired as the degree, specificity and stability of gene silencing are not always predictable . This necessitates screening and characterization of a large number of transgenic lines for the desired trait . By contrast, crispr / cas9 is guided to disrupt the reading frame thereby protein function of the target gene by dna editing . 2012), and both monoallelic and biallelic mutations are reported (bortesi and fischer, 2015). With biallelic editing, null mutations are readily obtained in primary transformants, though with varying efficiencies (brooks et al ., 2014; zhang et al ., 2014; zhou et al ., 2014, 2015), interestingly, the most efficient and consistent crispr / cas9 editing was reported in hybrid populus tremula x alba clone 717 - 1b4 (717), with biallelic mutations detected in all independent transgenic lines examined (zhou et al ., 2015). For woody perennials such as forest trees, fruit / nut trees and woody ornamentals with highly heterologous genomes and long generation times, crispr / cas9 affords a facile means to accelerate genetic improvement . The first application of crispr / cas9 genome editing in populus targeted the 4-coumarate: coa ligase (4cl) gene family involved in phenylpropanoid metabolism (zhou et al ., 2015). One of the genes, 4cl1 (potri.001g036900), has been extensively characterized for its involvement in lignin biosynthesis . Down - regulation of 4cl1 or its orthologs leads to reduced lignin content and altered lignin structure in a number of species (boerjan et al ., thus, crispr / cas9 editing of 4cl1 served as a proof - of - concept study, allowing an assessment of its efficacy in perturbing lignin biosynthesis relative to previous methods . We generated 36 independent transgenic lines, and amplicon - sequencing of randomly selected events confirmed biallelic mutations in all cases (zhou et al ., 2015). As frequently reported for crispr / cas9 genome editing, small indels, especially 1-bp modifications, were the predominant patterns and predicted to disrupt the reading frame . Consistent with this prediction, lignin content was reduced by 23% with a concomitant decrease of s / g lignin ratio by 30% in all transgenic plants (zhou et al ., 2015). Wood discoloration is a known side effect of lignin modification frequently observed in transgenic plants with suppressed lignin gene expression . However, the coloration patterns reported in those cases lack the uniformity we observed for the crispr / cas9 mutants . For example, antisense downregulation of 4cl1 in populus resulted in patchy wood discoloration in only 5 of 14 transgenic lines (voelker et al ., wood discoloration patterns of transgenic populus with down - regulated expression of cinnamoyl - coa reductase were highly variable among vegetatively propagated plants (van acker et al ., 2014), presumably due to the unstable nature of sense- or antisense - mediated post - transcriptional gene silencing . Rnai - mediated gene suppression is considered a more effective alternative, with improved trait stability (li et al ., 2008). For instance, rnai - silencing of 4-coumaroyl - coa 3-hydroxylase in transgenic populus resulted in lignin reductions among 9 independent events that ranged from no change to 1560% (coleman et al ., similarly, rnai silencing of 4cl in pinus led to wood discoloration in only 2 of 12 viable transgenic lines (wagner et al ., 2009). By contrast, the homogeneity of wood discoloration both within and across independent crispr / cas9-edited populus mutant lines is consistent with a null 4cl1, and resembles that of naturally occurring brown midrib mutants of maize and sorghum (sattler et al ., 2010). The results serve as testament to the superiority of crispr / cas9 over previous gene silencing methods . (2015) with permission . A second gene, 4cl2 (potri.019g049500) that is phylogenetically distinct from lignin - associated 4cls (chen et al ., 2014). Has long been suggested to participate in flavonoid biosynthesis, based solely on its preferential expression in epidermis and roots (harding et al ., 2002). Crispr / cas9 mutation of 4cl2 was therefore expected to yield functional evidence to substantiate this role . As with the 4cl1 case, biallelic mutations were observed in all independent transgenic lines surveyed by amplicon - sequencing (zhou et al ., the metabolic consequence was examined in roots where flavonoid - derived condensed tannins (cts) are known to accumulate to high levels (20% dry weight) in the experimental poplar genotype (chen et al ., 2014). Crispr / cas9 mutations of 4cl2 resulted in significantly reduced levels of condensed tannins in roots, providing reverse genetics evidence in support of class ii 4cl function in flavonoid biosynthesis . After our initial publication, successful crispr / cas9 editing was reported for populus tomentosa (fan et al ., 2015). That study targeted a phytoene desaturase (pds) gene for mutagenesis by multiplexing 4 grnas, and observed a 50% frequency of albino phenotypes . The lower mutation rates compared to our study may be attributed to gene redundancy and sequence heterozygosity that were not accounted for during grna design (see discussion below). Regardless, selected albino transgenic lines were sequence - confirmed to harbor biallelic mutations (fan et al ., 2015), providing additional support for the superior phenotypic uniformity of crispr / cas9 mutants . A major concern in genome editing is the specificity of the dna modification, as off - target cleavage due to non - specific crispr / cas9 activity may cause unintended mutations . This is especially true for plant genomes that are characterized by recurring episodes of whole - genome, segmental and tandem duplications . We assessed crispr / cas9 specificity in populus by analyzing its on - target versus off - target activities among duplicated genes . The grna for 4cl1 was designed to discriminate against the paralogous 4cl5 (potri.003g188500), with 3 mismatches in the target sequence and one mismatch in the protospacer adjacent motif (pam) (zhou et al ., 2015). By designing consensus primers flanking the target site, we were able to sequence both 4cl1 and 4cl5 amplicons simultaneously to assess the specificity of crispr / cas9 . A custom program, ageseq (analysis of genome editing by sequencing), was developed to facilitate variant detection (xue and tsai, 2015). No off - target cleavage in the 4cl5 locus was detected in 4cl1-edited lines, suggesting that crispr / cas9 editing in populus is highly specific (zhou et al ., 2015). Negative experiment, where mismatches between a 4cl5-grna, designed based on the p. trichocarpa reference genome (phytozome v3), and the corresponding sequence of p. tremula x alba 717 used for transformation abolished cleavage in all transgenic lines examined (zhou et al ., 2015). In this case, the mismatches were in the form of 2 single nucleotide polymorphisms (snps), one per allele, located near or in the pam . These results, together with the highly heterologous nature of woody perennials, suggest that off - target cleavage of crispr / cas9 is likely to be low in these species . The ability of crispr / cas9 to discriminate single - base differences makes it a powerful tool to investigate functional redundancy of highly homologous genes that are otherwise difficult to discern using gene silencing approaches . For instance, 4cl1 is the most abundantly expressed isoform in populus xylem, constituting> 85% of 4cl transcripts, with 4cl5 being a distant second, based on rna - seq data (swamy et al ., 2015). However, the crispr / cas9 knockout of 4cl1 reduced lignin content only by 23% (zhou et al ., 2015). Because the 4cl5 locus was unmodified, it is likely involved in the residual lignin biosynthesis of the 4cl1-knockout mutants . In the populus study that targeted pds, 4 grnas were designed based on one gene model (potri.014g148700) without considering the genome duplicate (potri.002g235200) that shares a high degree (93%) of coding sequence identity . While the genome sequence of p. tomentosa is not yet available, analysis of the p. trichocarpa and p. tremula x alba orthologs revealed different degrees of sequence variation between the 2 duplicates in the grna target regions . These differences (plus unknown allelic heterozygosity) likely contributed to the variation in the reported editing efficiencies of different grnas (fan et al ., 2015). Examples include designing multigene - targeting grnas based on conserved sequences of homologous genes (brooks et al ., 2014; jacobs et al ., 2015), or multiplexing individual grna cassettes (brooks et al ., 2014; fan et al ., 2015; zhang et al ., 2014 a recent study hijacked the trna processing machinery for efficient processing of up to 8, and potentially more, grnas from one single construct (xie et al ., the grna is positioned downstream of trna as a polycistronic gene with repeating trna - grna units for multiplexing (xie et al ., 2015). Because mature grnas are released following cleavage of the polycistronic transcript by the trna processing machinery, there is no sequence constraint at the 5 end of grnas (xie et al ., 2015). This greatly simplifies grna design, as any sequences upstream of pam (ngg) can be used (e.g., n20-ngg or other truncated lengths (fu et al ., 2014)), unlike with the standard design where grna is transcribed directly from the pol iii u6 or u3 promoter using a specific transcription initiation sequence (e.g., gn19-ngg or an19-ngg, respectively) (wang et al ., 2008). In some cases, grna stacking, either by re - transformation or via controlled crosses of primary transformants may be necessary in order to discern individual gene functions . As discussed above, numerous studies have exploited sequence polymorphisms for developing gene- or allele - specific grnas to achieve precision editing by crispr / cas9 . However, few studies have addressed the added complexity of sequence polymorphisms on crispr / cas9 genome editing, probably because the common plant models, such as arabidopsis, rice, soybean and tomato, are selfing species with homozygous genomes . We argue that sequence polymorphisms are of concern in genome editing of most flowering plants, especially woody perennials that are outcrossing in nature . This is supported by the high frequency of genic snps reported for trees, one per 60 bp in populus tremula (ingvarsson, 2005) or one per 16 - 33 bp in eucalyptus depending on the species (klheim et al ., 2009). A recent population genomics study of 544 populus trichocarpa individuals identified 17.9 million (m) snps by whole - genome resequencing (evans et al ., 2014). Using a similar approach with both resequencing and rna - seq data, we uncovered over 10 m snps from a single individual, p. tremula x alba clone 717 that is commonly used in populus transformation (xue et al ., 2015). Given the high levels of heterozygosity in tree species, the need to consider sequence polymorphisms in genome editing experiments cannot be over - emphasized . In fact, most published genome sequences are, by default, consensus sequences of their (pseudo)haploid genomes . Thus, while several web - based grna design programs are available for plants, such as crispr - plant (xie et al ., 2014). And crispr - p (lei et al ., 2014), their utility for outcrossing species is limited because the preloaded genome sequences lack biallelic (or multiploid) coverage . Furthermore, it is often the case as in populus that the species or genotype chosen for whole genome sequencing (e.g.,, p. trichocarpa nisqually-1) is different from that routinely used for genetic transformation (e.g., p. tremula x alba 717). To facilitate crispr / cas9 genome editing in 717, we constructed a custom 717 genome by substituting> 10 m sequence variants into the p. trichocarpa reference genome (xue et al ., 2015). We then assessed the impact of sequence polymorphisms on grna specificity for 1000 randomly selected genes . Grnas were designed based on the haploid genome of (1) p. trichocarpa or (2) 717, and then cross - checked for snps or indels in the 717 target sequences . From the first analysis, we found that more than 57% of the grnas designed based on the p. trichocarpa genome correspond to regions that harbor snps or indels in 717 (xue et al . The rates were slightly lower but nevertheless high (42 - 44%) in the second analysis when the custom 717 genome was used, owing to allelic variations (zhou et al ., 2015). Together, these data suggest that standard grna designs may suffer from a high probability of failure in crispr / cas9 genome editing, due to the frequent occurrence of snps / indels in outcrossers . The 717 sequences and associated blast and gene model query tools are available via aspendb (http://aspendb.uga.edu/s717). Tool is particularly useful for screening custom grnas or pcr primers against known snps / indels in the p. trichocarpa or the custom 717 genome (xue et al ., 2015). A set of pre - selected grnas with no known sequence variants for 38,509 populus genes is provided (zhou et al ., 2015). The modified, variant - sensitive pipeline for grna design, called aspen crispr designer, is also available at aspendb and can be applied to other species . For outcrossing species lacking a genomic variant database, several approaches can be applied to identify snps / indels in the gene(s) of interest prior to grna selection . Rna - seq data from the transformation genotype, when available, is an excellent source for high - confidence snp discovery . Provided that the gene of interest is reasonably expressed (e.g.,, with a high read coverage), manual inspection of candidate grnas against rna - seq alignments using programs such as the popular integrative genomics viewer (http://www.broadinstitute.org/igv) is usually effective to confirm their specificity . In the absence of deep - sequencing data, pcr primers (with or without degeneracy) can be designed to amplify the target gene coding region based on a closely - related reference genome, or on conserved amino acid sequences of orthologs as in conventional rt - pcr cloning (hu et al ., 1998). As pam (ngg)the only sequence constraint in grna design occurs frequently, it is not necessary to obtain full - length gene sequences . However, the use of multiple primer pairs is recommended to increase the likelihood of capturing both alleles . Direct sequencing of pcr products to identify sequence variants is an established practice in molecular ecology research, and several programs have been developed to decode overlapping sanger chromatograms (dmitriev and rakitov, 2008). For researchers with convenient access to high - throughput sequencing, sequence reads can then be processed by ageseq to search for potential variants in the target gene sequences (xue and tsai, 2015). Ageseq is available as a standalone program or a galaxy - compatible tool (https://toolshed.g2.bx.psu.edu) to support web - based data analysis . It should be noted that pcr amplification of noncoding sequences can be tricky due to the lower degree of sequence conservation (xue et al ., 2015). Thus, for crispr / cas9 applications that target promoter or other noncoding sequences (e.g., transcriptional activation or chromatin modification (sander and joung, 2014)), resequencing of the transformation clone for unbiased variant calling, as discussed above for populus 717 (xue et al ., 2015), is recommended . The populus study extended the ever growing list of plant species successfully genome - edited by crispr / cas9 (bortesi and fischer, 2015; kumar and jain, 2015) to woody perennials . Woody perennials suffer as experimental systems due to the lengthiness of genetic transformation and plant characterization . Many forest and horticultural tree species and genotypes are notoriously recalcitrant to tissue culture regeneration with low transformation efficiencies (litz and padilla, 2012; busov et al ., 2005). Limitations associated with other established technologies, such as incomplete silencing, position effects or epigenetic modifications of transgenes (matzke and matzke, 1998), further impede progress in transgenic tree research . Even for species with a facile transformation system, such as populus tremula x alba 717, the common practice of generating multiple independent events for preliminary screening prior to in - depth characterization of selected lines is resource - demanding and time - consuming (fig . 2). The ability of crispr / cas9 to generate null mutations in primary transformants, and the promise of phenotypic consistency among independent biallelic mutants as demonstrated in the populus study make a compelling argument that it may soon become acceptable to characterize only a few transgenic lines with sequence - verified mutations . High - throughput methods for mapping genome editing patterns (xue and tsai, 2015) and transgene (e.g., t - dna harboring cas9, grna and selectable marker) insertion sites (kanizay et al ., 2015) are expected to facilitate this effort . As depicted in figure 2, primary transformants with confirmed editing can be used directly for in - depth characterization when multiple independent events are available as biological replicates (zhou et al ., 2015). Even when only a few mutation events are obtained (for difficult - to - transform species or from suboptimal transformation trials), the ability to bypass rna - level screening and to obtain stable mutations still confers advantages over the gene - silencing approaches . Crispr / cas9 thus offers the enticing prospect of streamlined transgenic characterization and expedited large - scale functional characterization . Figure 2.schematic comparisons of experimental approaches involving gene silencing vs. crispr / cas9 genome editing . (a) for gene silencing by antisense, sense or rnai approaches, knowledge about the spatiotemporal expression pattern of the target gene is used to select an appropriate promoter for construct preparation . Expression knowledge is not necessary for crispr / cas9 editing at the dna level, and vectors with a pol iii promoter (e.g., u6 or u3) for grna expression and a cas9 under control of a constitutive (e.g., 35s) promoter are widely applicable . (b) following regeneration of putative transgenic plants, conventional screening involves pcr confirmation of transgenes followed by expression analysis to select events with desired (or maximum) levels of gene suppression . It is common to screen a large number of events (10) to identify a minimum of 3 for subsequent analysis . This process can be greatly simplified with crispr / cas9 mutants, as dna level analysis by amplicon sequencing can identify events with biallelic mutations for further analysis . (c) for species with a robust transformation system, multiple biallelic crispr / cas9 events can be used directly as biological replicates for in - depth characterization . In case of difficult - to - transform species for which few biallelic events may be obtained, micropropagation is necessary to obtain biological replicates before in - depth characterization, as is typically done using gene silencing . (a) for gene silencing by antisense, sense or rnai approaches, knowledge about the spatiotemporal expression pattern of the target gene is used to select an appropriate promoter for construct preparation . Expression knowledge is not necessary for crispr / cas9 editing at the dna level, and vectors with a pol iii promoter (e.g., u6 or u3) for grna expression and a cas9 under control of a constitutive (e.g., 35s) promoter are widely applicable . (b) following regeneration of putative transgenic plants, conventional screening involves pcr confirmation of transgenes followed by expression analysis to select events with desired (or maximum) levels of gene suppression . It is common to screen a large number of events (10) to identify a minimum of 3 for subsequent analysis . This process can be greatly simplified with crispr / cas9 mutants, as dna level analysis by amplicon sequencing can identify events with biallelic mutations for further analysis . (c) for species with a robust transformation system, multiple biallelic crispr / cas9 events can be used directly as biological replicates for in - depth characterization . In case of difficult - to - transform species for which few biallelic events may be obtained, micropropagation is necessary to obtain biological replicates before in - depth characterization, as is typically done using gene silencing . Biallelic mutations of the target gene(s) in conjunction with the hemizygous nature of transgene (e.g., t - dna) integration in primary transformants (t0) means that dna - edited but transgene - free progenies can be readily obtained from t1 segregants, as demonstrated in annual species (zhang et al . Trait stacking with early flowering induction is thus highly desirable, for example by over - expression of flowering locus t (ft) as demonstrated in prunus (plum), populus and eucalyptus (hoenicka et al ., 2014; another concern for outcrossing species is that the progenies will be different from the parents, which necessitates additional screening and selection . We note, however, that introgressing transgenic / mutant events into elite genotypes is an established practice in crop and tree breeding programs, and hence not unique to crispr / cas9-edited mutants . A well - known tree example is the loblolly pine (pinus taeda) clone 756 that harbors a naturally occurring null allele of cinnamoyl alcohol dehydrogenase involved in lignin biosynthesis, and that is extensively used as a parent in several breeding programs in the us (gill et al ., 2003). Another example is early - flowering ft - plum that serves as a parent in the fasttrack breeding program to expedite development of improved commercial cultivars (callahan et al ., various molecular and genomics tools are available to facilitate progeny selection in transgenic breeding programs . Recently, virus - based systems have been explored for transient expression of genome - engineering reagents (ali et al . However, such systems still utilize t - dna to express the repurposed viral genome components, and the resulting plants are not transgene - free without crossing . These methods also depend on agroinfiltration to deliver the vector into plant cells, which unfortunately is not applicable to most woody species with an open minor vein structure in their leaves (gamalei, 1989). Clearly, development of innovative methods will be necessary for facile removal of t - dna from the genome - edited plants without crossing . Regardless, null segregants of crop cultivars derived from genome editing are indistinguishable from naturally occurring or chemically - induced mutants, and may therefore be exempt from gmo regulation in countries that adhere to product - based regulations (voytas and gao, 2014). This is expected to significantly reduce the timeline and financial burden associated with developing new crop varieties, thereby encouraging adoption of crispr / cas9-based genome editing for the forest products, fruit / tree nuts, and woody ornamental industries . The research in the tsai laboratory discussed in this article was supported by the department of energy, office of biological and environmental research (grant no . De - sc0008470), the department of agriculture, national institute of food and agriculture (grant no . 2015 - 67013 - 22812), and by the georgia research alliance - hank haynes forest biotechnology endowment.

Like co@fe3o4, which recently underwent extensive study to elucidate both the oxidative stability of the core and the phase of the shell, fept@fe3o4 core shell nanoparticles can be used as building blocks to form nanocomposites with enhanced magnetic properties with the potential for novel applications . These include magnetic data storage, catalysis, and targeted drug delivery . In terms of nanomagnetics specifically, our hope is that the presence of an inert oxide shell may function to inhibit agglomeration of the cores upon annealing; this is a necessary step in creating an ordered l10 fept bimetallic structure with a higher magnetic coercivity . The determination of the core composition is important for tailoring their synthesis in order to ultimately achieve the desired 50:50 fept alloy composition . A solution of core shell particles was drop - cast onto a 3 mm holey carbon copper grid . Edx data were acquired using an fei osiris tem equipped with a high brightness schottky x - feg gun and a super - x edx system comprising four silicon drift detectors, each approximately 30 mm in area and arranged symmetrically around an optic axis to achieve a collection solid angle of 0.9 sr . Edx data were collected in the form of spectrum images, in which a focused electron probe was scanned in a raster across a region of interest in the scanning tem (stem). At each point in the scan, structural information was obtained from the electron scattering incident on a high angle annular dark field (haadf) detector, and simultaneously, an edx spectrum was obtained by collecting x - rays emitted from the local volume probed by the electron beam . The resulting edx spectrum image was a three - dimensional data set whose (x, y) axes correspond to the position of the probe and whose z axis corresponds to the energy of the detected x - ray . Spectrum images were acquired with a probe current of approximately 0.7 na, an acceleration voltage of 200 kv, a spatial sampling of between 0.5 and 1 nm / pixel and 50100 ms / pixel dwell times . X - ray intensities were obtained by fitting a model of the edx spectra to the experimental data using weighted least - squares and atomic fractions were quantified from intensities using the cliff lorimer quantification . The edx model and the quantification were implemented in hyperspy and will be available in future releases of the software . Figure 1a displays a haadf stem image obtained during the acquisition of a spectrum image enclosing a cluster of 13 co@fe3o4 nanoparticles . Although the particle morphologies are seen to vary slightly from one particle to another, the majority of particles have a round core approximately 20 nm in diameter surrounded by a thin shell approximately 5 nm in thickness . The edx elemental maps for cobalt, iron, and oxygen (figure 1b d), obtained by integration of the element s background - subtracted k - line x - ray peak, show that individual particles are comprised of a cobalt core surrounded by a shell composed of iron and oxygen, as expected . The largest particle in the lower left region of the map appears to have more iron in the core compared to the smaller particles . These conventional edx element maps show the location of the various elements, but the composition of the particle core, for example, cannot be determined by elemental mapping due to the presence of the shell above and below the core in projection . Elemental maps of (b) cobalt, (c) iron, and (d) oxygen display the location of the various elements with respect to the particle morphology (scale bar = 50 nm, greyscale = x - ray counts). The edx spectrum image data of the co@fe3o4 nanoparticle cluster shown in figure 1a was subsequently processed using bss methods in hyperspy . First, the spectral dimension in the data set was binned by four from 5 ev / channel to 20 ev / channel in order to increase the number of counts per channel . Next, we note that the binning step is necessary in order to optimize the accuracy of the variance stabilization channel . The first three principal components, pc#0, pc#1, and pc#2, exhibited significantly greater variance than the remaining components (figure 2a), which suggests that there are only three phases present in the sample . That being the case, those three pca components should be a linear combination of the spectra and distribution maps of those phases, but the mixing matrix is unknown . Next, we compute numerically the first derivative of the pca spectral components in order to diminish the correlation caused by the edx background, and we use fastica to estimate the mixing matrix and compute the independent components (ics) ic#0, ic#1, and ic#2 (figure 2b) and their distribution maps (figure 2c e) from the pca results . Component independence is a much more stringent property than uncorrelatedness imposed by pca . If we disregard the small copper peaks contained in all the independent components, likely originating from the copper support mesh, we see that ic#0 contains cobalt x - ray peaks, ic#1 iron and oxygen peaks, and ic#2 a carbon peak . The three ics appear to belong to the three phases present in the originally scanned area: the core, shell, and supporting film . This hypothesis is explored further in the next section . At this point, however, it is important to note that, unlike in the conventional edx mapping shown in figure 1, no elements were selected prior to performing ica, and thus, the analysis is free of external bias, except for the choice of the number of components . Result of bss by pca and ica of an edx si of a co@fe3o4 core (b) corresponding independent component spectra contain the expected x - ray lines for the elements present . Independent component maps (c e) show that (c) ic#0 is concentrated in the nanoparticle cores, (d) ic#1 in the shells, and (e) ic#2 everywhere on the carbon supporting film (scale = 50 nm, greyscale = normalized weighting). We now move on to the analysis of a second cluster of particles comprised of a bimetallic iron / platinum core surrounded by an iron oxide shell . A crystalline core surrounded by a polycrystalline oxide shell is observed in the representative high - resolution stem haadf image shown in figure 3 . The particle morphologies were found to have a mean core diameter of approximately 3.3 nm and mean shell thickness of approximately 1.7 nm (figure 4a). Also visible were pure iron oxide particles (in the lower right - hand corner of figure 4a). Ica of a cluster of bimetallic platinum / iron nanoparticle seeds coated by fe3o4 shells . (b) scree plot of the first 50 principal components showing the first three components lying above the noise . (c e) element maps of (c) platinum, (d) iron, and (e) oxygen . (f h). The ic maps (f) ic#0, (g) ic#1, and (h) ic#2 and (i) the corresponding ic spectra . From the selected element maps (figure 4c e), it is clear that, with the exception of the two particles in the lower right - hand corner, the particles are comprised of a platinum rich core surrounded by an iron oxide shell . However, it is not clear from the maps alone whether iron is present in the core . By conventional elemental mapping, one cannot tell whether the particles contain a pure platinum core surrounded by an iron oxide shell or whether iron is alloyed with platinum to form a bimetallic core . We now address these questions by performing bss on the same edx spectrum image using the same procedure detailed before . Once again, by inspecting the scree plot (figure 4b), we find that the sample consists of three phases . The spatial distribution of ic#0 is concentrated in the particle cores, ic#1 in a shell around the cores, and ic#2 is approximately uniformly distributed over the map (figure 4f h). If we again disregard the spurious copper peak, ic#0 contains iron and platinum x - ray peaks, ic#1 iron and oxygen peaks, and ic#2 a single carbon peak (figure 4i). From this analysis it appears that the ica components represent the different phases present in the edx spectrum image; ic#0, ic#1, and ic#2 genuinely represent the bimetallic fept cores, iron oxide shells and the carbon support, respectively . In order to verify the accuracy of the ica results, we evaluated whether ic#0 represented the true composition of the core by analyzing bare fept seed particle clusters obtained from the same chemical synthesis but extracted prior to the shell addition step . Being made from the same synthesis, the composition of the bimetallic seed particles are expected to match to the composition of the bimetallic cores in the core shell particles analyzed in figure 4 . A total of 12 edx sis were acquired in order to capture and analyze multiple fept bimetallic seed clusters . The segmentation of one of the edx sis is shown in figure 5a, b . To accurately extract the intensity of the fe k and pt l peaks, a model composed of one gaussian per x - ray line and a background based on kramers and small expressions as developed elsewhere was used, as shown in green in figure 5c . The only free parameters of the model were the area of the gaussian and the height of the background, which was negligible . The mean reduced over the fit of all particles was 1.01, indicating that the discrepancies between the model and the data are in accordance with the poisson noise variance . The intensities of fe k and pt l peaks in the fitted model were quantified using the cliff lorimer method . The obtained compositions are plotted for each particle in figure 5d along with the fitting error estimated from poisson statistics . The raw data were decomposed using pca on all fept seed data and the first three components were retained for noise reduction . The 103 individual fept bimetallic seeds analyzed were found to have a mean composition of 82.0 at .% pt, which lies well within one standard deviation of the average bimetallic seed composition . The data points in figure 5d are displayed in order of ascending particle size . As such, the calculated compositions on the right - hand side tend to have a smaller error bar on account of the signal noise ratio being higher for larger particles . The compositions of the larger seed particles are also closer to the mean composition and to the composition of ic#0, confirming the homogeneity of the composition and the validity of the bss analysis . Summary of the composition of 103 bare fept bimetallic nanoparticles extracted from a synthesis prior to the shell addition step . (b) segmentation of the edx spectrum image prior to quantification of each particle . (c) fitting of the edx spectrum from a single seed (circled in (b)) to a model spectrum to determine the fe k and pt l peak intensities . (d) particle seed compositions obtained by quantifying the fitted intensities from 103 different particles (error bars = 1 standard deviation). A comparison of raw edx spectra extracted from fept bimetallic seed particles and from pure fe3o4 particles with ic#0 and ic#1, respectively, is provided in figure 6 . In both cases, the ics were scaled by a constant to obtain a best fit to the raw spectra . Despite the strong overall agreement in each case, the carbon peak difference in both cases is caused by the separation of carbon into a different component (ic#2). The difference in the shell cu peaks are not due to a compositional difference as the cu signal is spurious in origin . The iron oxide particle spectrum also contains small silicon and sulfur peaks which likely originate from residue on the carbon film . The difference in the pt m core peak may be due to the attenuation of pt m x - rays in the shell of the core shell nanoparticles and in nearby particles along the trajectory to the detector . The strong overall similarity between the raw and ic spectra provide direct evidence showing that the spectral components extracted by ica from the core shell spectrum image data are strongly representative of the buried core, surrounding shell, and carbon support compositions . Comparison between raw edx spectra extracted from a fept bimetallic seed particle (top) and from an iron oxide particle (bottom) with ic#0 and ic#1, respectively . The fept seed and fe3o4 x when analyzing beam - sensitive materials, the main limitation to the accuracy of the bss analysis method that we propose is the intensity of the edx signal achievable without inducing significant sample damage . In our case, despite the use of a high efficiency edx system, the number of collected x - rays is low . In order to avoid the artifacts that arise when using the variance - stabilizing transformation out of its domain of application, we have binned the data by four in the spectral dimension from 5 ev / channel to 20 ev / channel . Given that the resolution of our edx detector is approximately 130 ev at mn k and that there were no overlapping x - ray lines, in our case, the increase in the number of counts per channel (and hence in the accuracy of the analysis) comes without significant resolution loss and, therefore, should not have any adverse effect in the analysis . A blind source separation method based on pca and ica has been applied to the analysis of edx spectrum images of core the analysis has accurately determined the number of phases in the analyzed volume (core, shell, and supporting film) as well as their spectra and distribution maps . We have confirmed the accuracy of the analysis by comparing the calculated spectra from the platinum iron core and the iron oxide shell to those obtained from these structures in isolation, and the excellent agreement suggests that bss, therefore, can be used to accurately analyze edx data . The use of ica on edx spectrum image data promises to be a powerful technique for extracting buried compositions at the nanoscale in a variety of materials, and further testing on the method s applicability to different systems is now being initiated.

Ionic calcium (ca) controls multiple cellular signaling processes in all eukaryotic cells, including proliferation, gene expression, and neurotransmitter release . Ca - binding proteins such as calmodulin play a pivotal role in ca signal transmission and amplification . Increases in the concentration of intracellular ca activate specific protein targets, among which the ca / calmodulin - dependent protein kinase ii (camkii) is a critical signal mediator . In response to an increase in intracellular ca, thr-286 of camkii the coupling of ca - calmodulin to one of the camkii subunits allows for the phosphorylation of an adjacent subunit at thr . Calmodulin trapping, confers ca - calmodulin - independent kinase activity to the complex and thus prolongs the ca signal . Thus, calmodulin trapping represents a molecular mechanism of memory, which is defined as the capacity to acquire, store (consolidate), and retrieve (evocate) information . Camkii is a major synaptic protein that is activated during the induction of long - term potentiation (ltp) by ca influx through n - methyl - d - aspartate (nmda) receptors . Calmodulin trapping allows camkii to remain activated long after the initial ca signal has dissipated, suggesting that camkii is a memory molecule crucial for ltp . Consistent with this notion, camkii - null mice present with impaired memory formation, and camkii is essential for genesis and maintenance of ltp in postsynaptic neurons . Following presynaptic stimulation, camkii is activated in postsynaptic neurons, which creates a physiological imprint of the initial ca signal, and increases translocation of nmda receptors to the plasma membrane . Because of its capacity to remain activated long after the initial pulse of ca signaling, camkii perpetuates ca effects and modulates gene expression and the epigenetic profile of postsynaptic neurons . Camkii also participates in glucose - stimulated insulin secretion (gsis), as multiple insulin secretagogues increase camkii activity . In the perfused rat pancreas, the dynamics of camkii activation correlate with the amplitude of gsis, and camkii activation is temporally associated with insulin secretion . Camkii is essential for appropriate gsis and is involved in several steps of this process, including the synthesis of insulin granules, the modulation of cytoplasmic content of atp, and the activation of synapsin . Importantly, camkii also regulates transcription factors central for - cell function such as creb and mafa . Here, we investigated whether pancreatic - cells, like neurons, acquire and store the information contained in calcium pulses as a form of metabolic memory . Indeed, we find that - cells retain memory of prior activation, and describe the molecular mechanism that contributes to this memory . Min6 insulinoma cells or mouse or human pancreatic islets were distributed into three groups: control, pulse, and pulse + kn93 (10 m). Initially, all groups were maintained in the same medium with 5.6 mm glucose for 24 h. after this acclimation period, the control group was exposed to 3-mm glucose for 24 h, while the pulse and pulse + kn93 (10 m) groups were exposed to 30 mm of glucose for four 1-h periods intercalated with 7-hour periods of 3 mm glucose . Kn93 (a camkii inhibitor) at 10 m was only present during the 30 mm glucose pulses . All groups were then maintained at 3 mm glucose for a 24-h consolidation period . Insulin secretion of min6 cells and mouse pancreatic islets was conducted following standard procedures, and insulin was measured by ria . One hundred and fifty islets were handpicked under a light microscope and pre - incubated for 1 h in krbb (krebs bicarbonate buffer) containing 0.3 the medium was discarded and the islets incubated for an additional hour in 500-l krbb containing 2.8 or 16.8 mm glucose . Subsequently, the supernatant was collected to evaluate insulin secretion by ria . For perifusion assays, 150 treated human islets were placed into a perifusion chamber (millipore, billerica, ma, usa). A computer - controlled fast - performance hplc system allowed programmable rates of flow and concentrations of the appropriate solutions held in a 37 c water bath . Islets were perfused for 80 min with krbb in the absence of glucose, followed by krbb with increasing concentrations of glucose (glucose was ramped from 0 to 30 mm at 0.75 mm / min). At the end of each experiment, islets were tested for maximal insulin secretion by adding 30 mm kcl to the perifusate . Samples were collected at 2 ml / min and insulin content was determined by using ria . Statistical analyses were performed using student's t - test or a two - way anova with a bonferroni's posttest, as required . Statistical significance was set at p <0.05 . In order to investigate metabolic memory of insulin - producing - cells, we first had to develop a paradigm in which such memory was evident . As detailed below, we established that exposure of cultured min6 - cells to four 1-h pulses of high glucose, interspersed with 7-h intervals of low glucose and thus mimicking postprandial glucose spikes in vivo, were able to elicit robust metabolic memory . The detailed experimental paradigm is outlined in figure 1a . As shown in figure 1b, min6 - cells exposed to the glucose pulse regimen showed higher insulin secretion both when exposed to low (2.8 mm) or high (16.8 mm) glucose than - cells, which had been cultured continuously at low glucose . This increase in insulin secretion was accompanied by a significant rise in the levels of phosphorylated camkii, consistent with the notion that camkii might be a molecular mediator of this metabolic memory (figure 1c and d). Importantly, both the increases in basal and stimulated insulin secretion in the glucose pulse group were abolished by kn93 (figure 1b), a specific camkii inhibitor . Next, we evaluated the kinetics of the - cell response to acute glucose exposure, and observed that the pulse group secreted more insulin at basal (time zero) and stimulatory concentrations of glucose (16.7 mm) between 5 and 60 min following stimulation (figure 1e). This effect persisted even after glucose concentrations were returned to 2.8 mm . In parallel, the phosphorylation of camkii was higher in the glucose pulse group at both glucose concentrations, and remained elevated even after the glucose concentration was reduced to 2.8 mm (figure 1f). Thus, min6 - cells had acquired metabolic memory of prior exposure to glucose pulses, which they retained through a 24-h consolidation period . It was important to determine if metabolic memory is confined to min6 insulinoma cells, or if it is also a property of primary human - cells . Therefore, we cultured human islets from multiple non - diabetic deceased organ donors (see table 1 for donor information) and exposed them to pulses of 30 mm glucose in the same paradigm as described above . Pulse treatment increased insulin secretion at basal (2.8 mm) and stimulatory (16.8 mm) glucose concentrations (figure 2a). As was the case in min6 - cells, the pulse group showed increased camkii phosphorylation at both basal and stimulatory glucose concentrations (figure 2b). Next, we determined the human islet response to a glucose challenge in the islet perifusion assay . Islets from the glucose pulse group showed both increased first and second phase insulin secretion compared with islets from the control group (figure 2c and d). As before, these effects were abolished by the camkii inhibitor kn93 (figure 2a d). We also tested metabolic memory of - cells using isolated mouse pancreatic islets, with similar outcome (extended data figure 1). We also found, using live cell calcium imaging of mouse islets, that the increases in cytoplasmic calcium levels following acute glucose stimulation, representing the penultimate step in insulin secretion, are significantly higher in the pulse group compared with controls (extended data figure 1c). To investigate the molecular mechanisms by which the glucose pulse paradigm induces memory in - cells, we evaluated the expression of essential mediators of the insulin secretory response . The glucose pulse paradigm induced expression of glucokinase (gck), the main glucose sensor of - cells (figure 3a), the voltage - gated ca channel (cav1.2) (figure 3b), snap25, an essential component of the exocytotic machinery (figure 3c), and mafa, a key transcription factor of the insulin gene and the mature - cell phenotype in general (figure 3d). All the effects of the pulsed glucose paradigm on protein levels were reduced or abolished by treatment with kn93 during the high glucose exposure only . Finally, we asked if metabolic memory of - cells is dependent on glucose metabolism itself, or if this memory can be produced by membrane depolarization alone . To this end, we treated human islets with pulses of 30 mm kcl, which causes membrane depolarization, opening of voltage - gated ca channels, and insulin secretion without a prior increase in intracellular atp levels . As shown in figure 3e, depolarization by potassium chloride during the acquisition period produced similar, if somewhat lower, increases in pcamkii and psynapsin levels 24 h after the last kcl pulse . Thus, increases in intracellular calcium elicited by membrane depolarization are sufficient to activate camkii and produce metabolic memory . Total camkii protein levels were not affected by any of the treatments used (data not shown). Glucose - stimulated insulin secretion is a complex process that translates glycolytic flux and elevated atp production to increased cytoplasmic ca levels and finally fusion of insulin granules with the plasma membrane . This process is accompanied by increased phosphorylation of camkii (extended data figure 2), a process now shown to be part of the establishment of metabolic memory in - cells . Cerasi and colleagues had previously reported time - dependent potentiation of insulin secretion, in that exposure of rat islets to high glucose (27.7 mm) up to 60 min prior to the test stimulation with glucose increased subsequent insulin secretion . However, these studies focused on analyzing acute effects of high glucose pre - treatment, and did not yet investigate the molecular mechanism underlying the phenomenon . Here, we demonstrate that human and mouse pancreatic - cells are able to acquire, consolidate, and retrieve information, induced by high glucose exposure, and determined that this ability is dependent on camkii . In conclusion, we have shown here that, like neurons, human and mouse - cells are able to acquire, store, and retrieve information in a process similar to neuronal ltp . This process, presented schematically in figure 4, is dependent on the activation of camkii, as is the case for neuronal memory . These findings provide further evidence that the similarity of neurons and - cells on the transcriptome and epigenome level is not accidental, but an important aspect of the biology of these embryologically distinct cell types . Metabolic memory of - cells likely represents a useful evolutionary adaptation to variation in food availability . As periods of food abundance in nature can vary dramatically in length, metabolic memory of repeated carbohydrate loads in - cells ensures higher insulin secretion and thus more efficient uptake of excess glucose into skeletal muscle and adipose tissue for long - term storage, contributing to the adaptation to periods of starvation . Given the pivotal role of altered insulin secretion in metabolic disorders, future work will need to establish to what extent metabolic memory of pancreatic - cells contributes to the pathophysiology of these diseases.

Health is not only related to the absence of the disease, therefore we need to conceptualize and operationalize what health is . Increasingly, we have come to understand that information about functional status is needed in order to appreciate the full picture regarding the health of an individual or a population . An individual's health fundamentally includes their capacity to carry out the full range of actions, activities and tasks required to fully engage in all areas of human life . The health state of a person can be described in terms of capacity to carry out a set of tasks or actions . In addition, the health state also includes changes in body functions and/or structures arising from a health condition . The impact of the health state on a person's life can be understood by measuring performance of tasks and actions in the person's real - life or actual environment . The full picture of the health experience can further be appreciated by taking into cognizance the value that people place on levels of functioning in given domains in association with a health condition . Plainly, the concept of functional status is integral to health and its achievement . Two individuals with identical diagnoses may have utterly different levels of functioning that determine their actual health status . Without fsi, our picture of the health of an individual, or a population, is flawed and incomplete . Fsi has, of course, long been collected in various ways and used clinically, especially in rehabilitative medicine; physical, occupational and speech and language therapy; and in nursing home and home care settings . Fsi is essential for needs assessment as well as the development and monitoring of rehabilitative interventions to restore or maintain functions . It is also essential in this area of health care because the aim of therapy is to assist patients in maximizing their capacities to perform activities needed for their lives . Although no one doubts that restoring functioning is restoring health (the ultimate purpose of all forms of health care) some clinicians, focusing exclusively on acute - care needs, do not see the need to collect or utilize fsi . In most countries with a sophisticated health administrative data collection and utilization infrastructure, a wide variety of information what is often missing is information that would link diagnosis and treatment with health outcomes that are fully meaningful to the patient's life, namely information about the presence of decrements in capacity to carry out tasks and actions in areas of life as well as how these decrements play out in the person's actual, real - life environment (deyo and patrick, 1989; lubetkin et al ., 2003). There is growing recognition that there is a gap in health administrative records: the failure to collect or disseminate fsi across all health care settings . Unless fsi becomes an essential part of administrative records, the potential value of these data will be lost, not merely to clinicians, but to health administrators concerned about management and quality of care issues, health researchers, and public health agencies . This insight is clearly expressed in a report by the national committee on vital and health statistics (ncvhs) (2001): without functional status information, the researchers, policymakers, and others who are already using administrative data have at best a rough idea of how people, individually and collectively, are doing and at worst they are making erroneous assumptions and decisions . The report outlines in some detail the benefits of routinely collecting fsi across the entire health care delivery system and throughout all care settings . Fsi can serve management needs of all the stakeholders in the health care system clinicians, providers, payers, patients, and government regulatory bodies . This is true especially with respect to evaluating outcomes, comparing treatment modalities, and predicting and managing costs . This links directly to debates of modes of service provision, single or multiple payer, managed care, fee - for - service, or some hybrid mixture . The policy and research applications of fsi are evident for local health management and quality control, and in the broader arena of public health . Policy decisions about priorities must be made at the level of individual clinics or hospitals, local or regional health care agencies, or at the level of government planning and budgeting . Given the importance of getting the complete picture of health outcomes, fsi is an essential input into evidence - based policy decisionmaking . Researchers in all areas of health and social policy, at all levels, need valid and reliable data about functional status in order to make informed decisions . For example, it is a matter of debate whether, as the world's population lives longer and ages, they will be unhealthy and pose a greater burden on health systems . There is some evidence suggesting that elderly persons today are functioning at higher levels than before . Without reliable information on levels of functioning, this debate would be unresolvable because it would not be possible to detect functional status, since the disease morbidity may not have changed very much . Compression of morbidity occurs when disability or decrement in functioning is postponed more than longevity is extended, as for example with the effects of exercise or better eating habits . The direct test of compression (or extension) of morbidity depends on the effects of reduced health risks on cumulative lifetime disability (fries, 1980, 2001; vita et al .,, fsi is a crucial element for the description of health states and quantification of overall health status in individuals that can be aggregated to a summary measure of population health . At the who, the use of fsi is in this area, in particular, because this data (collected by the world health survey now in the field in more than 70 countries) feeds into ongoing endeavors to determine levels and distributions of health this survey would be inconceivable without information on health outcomes that describe health on multiple dimensions in terms of levels of functioning in a parsimonious set of domains . It is commonly known that the demographic trends toward an older population, at least in developed counties, will create unprecedented burdens on all age - sensitive social policies, such as social security and other pensions, retirement, unemployment, and long - term care . Aging, according to a recent organization for economic cooperation and development (oecd) (2001) report, is the principal factor currently driving pension spending costs . Since age - sensitive social programming constitutes between 40 and 60 percent of total public spending, the impact of aging is considerable . To comprehend the nature and magnitude of its social impact, those responsible for policies from transportation and housing to employment and taxation, will need reliable data on functional status and how it plays out in the lives of the aging population . For fsi to be available for this wide variety of uses, however, it must be routinely and consistently collected across the entire health care delivery system, preferably in some electronic format . Nonetheless, before contemplating the systemwide changes required to collect fsi, a classification that provides a common language and framework to describe the universe of functioning and disability is required . In order to complement the classification scheme, a comprehensive coding system that creates consistent and comparable data across all settings of care and a method of routinely capturing and disseminating these data (in a mode and manner consistent with social interests in preserving privacy) linked to measurement tools for clinical and related encounters the foundation of a new structure for collecting fsi is, therefore, a standard classification and coding system that will make it feasible for fsi to be included in administrative data . As the ncvhs report stated: while the international classification of diseases (icd) has served us well for more than a century in characterizing diagnoses, it is now time to complement it with a parallel system for characterizing functional status . Although the committee argued that more research, analysis, testing, and demonstration projects are required before final recommendations can be made, it concluded that: the concepts and conceptual framework of the icf have promise as a code set for reporting functional status information in administrative records and computerized medical records . In the committee's view, the icf is the only existing classification system that could be used to code functional status across the age span . In this article, we want to briefly describe the extensive international developmental process that lead to the revision of the original international classification of impairments, disabilities and handicaps (icidh) (world health organization, 1980) and produced the icf . We also want to describe the basic principles and structure of the icf, in particular, to show its value in the context of collecting fsi for administrative records . The primary mandate of who is the production and dissemination of reliable and timely information about the health of populations . Who's 1947 constitution requires that: each member shall provide statistical and epidemiological reports in a manner to be determined by the health assembly . Countries have long reported causes of death or mortality statistics based on who's (1992) international statistical classification of diseases and related health problems (icd-10). Though useful for calculating life expectancy for different countries, however, who recognized that these data did not capture the overall health status of living populations . Missing was information about non - fatal health outcomes, i.e., functioning and disability across all areas of life . To meet this need, who (1980) issued a tool for the classification of the consequences of disease, namely the icidh . A considerable academic literature built up around clinical and other uses of the icidh, but much of this literature was critical of the underlying model of disability . Responding to these critiques and an international call for an updated version, who launched a revision process in 1993 to address what many viewed as an urgent international need for a framework for measuring and reporting the health as functional status at both individual and population levels . Over the next 10 years, who's international collaborating centers and governmental and non - governmental organizations, including groups representing persons with disabilities, engaged in the systematic revision of the icidh . From an exhaustive literature search of existing classifications and assessment tools, the who revision team developed a 3,000-plus item pool of potential classification domain names for areas of human functioning at the body, person, and social levels . All efforts were made to ensure that the icidh-2, as it was initially named, would be a suitable classification for all domains of functioning associated with both physical and mental health conditions . Adopting the strategy of computer software development, alpha and beta drafts were prepared from 1996 forward . The original 1980 icidh had only been approved for field - trial purposes . In light of that, the who team felt for icidh-2 to have the necessary credibility and legitimacy to serve as the international standard language of health and functioning, that the revision process should include several years of field trials and other tests . The first phase of field trials concentrated on the cross - cultural and linguistic applicability of the model and classificatory structure and language of the icidh-2 . The intent of this phase of field trials was to establish the conceptual and functional equivalence of the items contained within the classification . Stn et al. (1999a, b; 2000) provide the rationale for the methodologies and presentation and analysis of the 15-country field trials . These results fed into further international collaboration in which the who team relied on a global network of who collaborating centers, non - governmental organizations, disability groups, and individual experts and key informants . The next revision phase began in 1999 when a series of expert drafting teams were assembled in geneva to produce the beta 2 draft . This draft was used for the second round of international field trials, these focusing on questions of reliability, utility, and feasibility of use . Once the results of these tests were collected and analyzed, a pre - final draft was produced in early fall 2000 as a result of an intensive editing process grounded in the expert input being received from around the world . The icidh-2, unlike its predecessor, was from the outset developed in multiple languages, primarily to identify and respond to cross - cultural and linguistic differences that might affect the usefulness of the classification . The collaborating centers and others provided constant input at this stage as the language and classification structures were redrafted and refined in multiple iterations . The draft was put on the internet for comment from a wide range of individuals, including both providers and consumers . After presentation before the executive board in december 2000, the classification was put on the agenda of the fifty - fourth world health assembly and renamed the icf . The new title reflected the philosophy of moving beyond the consequence of disease approach and highlighted functioning as a component of health . In may 2001, it was unanimously endorsed, member states were urged to use the icf in their research, surveillance and reporting as appropriate . With its approval, the icf became a member of the who family of international classifications . Whereas icd-10 provides the codes for mortality and morbidity, icf provides the codes to describe the complete range of functional states that capture the complete experience of health . The icd-10 and icf are, therefore, complementary and who encourages users to utilize both together, wherever applicable . This will ensure a more meaningful and complete picture of the health of people or populations . Soon after its official release, who's director general, gro harlem bruntland, announced that the icf is who's framework for measuring health and disability at both the individual and population levels . Who has already implemented icf as the basis for its extensive world health survey program, demonstrating its use as a global and universal tool . To improve health, tools are needed to measure health, and in particular to measure the changes in health brought about by interventions . Icf is the ruler with which we will take precise measurements of health and disability . (brundtland, 2002 .) From the public health perspective, the usefulness of icf goes beyond that of the measuring of population health and the effectiveness of internationally coordinated interventions funded by initiatives, such as the global fund to fight aids, tuberculosis and malaria . In addition, with the icf as their framework, countries will be able to identify social factors such as education, transportation, or housing, both as determinants of health, and social factors influenced by improvements in health . Making these links will further support the relationship between health and economic development . In short, we have in the shape of a little red book, an extraordinarily versatile tool a swiss army knife for health ministries, researchers and decision - makers . (brundtland, 2002 .) Undoubtedly the primary reason that icf can plausibly claim to be a universal tool for classifying states of functioning and disability is that the underlying model of the icf reflects our best understanding of the complex phenomena of functioning and disability in a manner that is, to the greatest extent possible, theory - neutral and therefore compatible with whichever theoretical account of how disability arises, at the individual and population levels, that evidence may confirm . It is the conceptual basis for the definition, measurement, and policy formulations for all aspects of disability . A paradigmatic shift in the thinking with regard to disability that is captured in the icf is the stress placed on health and levels of functioning . Heretofore, disability has been construed as an all or none phenomenon: a distinct category to which an individual either belonged or not . The icf, on the other hand, presents disability as a continuum, relevant to the lives of all people to different degrees and at different times in their lives . Disability is not something that happens only to a minority of humanity, it is a common (indeed natural) feature of the human condition . The icf is for all people, not just people traditionally referred to as disabled and isolated as a separate group . Icf thus mainstreams the experience of disability and recognizes it as a universal human experience . By shifting the focus from cause to the full range of lived experiences, it places all health conditions on an equal footing, allowing them to be compared using a common metric the ruler of health and disability . From emphasizing people's disabilities, and labeling people as disabled, we now focus on the level of health and functional capacity of all people . Decrements in functioning may be the result of decrements in intrinsic capacity or problems with body functions or structures; or they can result from features of the person's physical, human - built or social environment that lead to problems in performance over and above decrements in capacity . Very likely, decrements in functioning are the result of both processes . Yet, the extent to which intrinsic decrements in capacity or environmental factors are the cause is not a matter that can be determined a priori . Moreover, icf is grounded in the principle of universality, namely that functioning and disability are applicable to all people, irrespective of health condition, and in particular that disability or decrement in functioning at one or more levels is not the mark of a specific minority class of people, but is a feature of the human condition, which is, epidemiologically speaking, over the lifespan, a universal phenomena . In addition, icf is committed to the principle of parity, which states that the functional status is not determined by background etiology, and in particular by whether one has a physical rather than mental health condition . Much time, effort, and international collaboration has gone into the development of the icf . It is no longer plausible to insist that the icf is a medical classification of people with disability, that it reduces all issues of functional status to underlying medical conditions, that it ignores the often salient role of the physical and social environment in the creation of restrictions of participation experienced by persons with functional problems . The revision process has produced a classification that has already stood up to rigorous tests of validity, reliability, and cross - cultural applicability . It is, as the ncvhs has concluded, the only existing classification system that could be used to code functional status across the age span . We now turn to the structure of icf as a classification system, in part to show why the committee has correctly assessed the value of the icf as a coding system for functional status, suitable for use in administrative records . The model that informs icf, portrays functioning and decrements in functioning, or disability, as a dynamic interaction between health conditions (diseases, disorders, and injuries) and contextual factors . Contextual factors include environmental factors, that is, all aspects of the physical, human - built, social, and attitudinal environment that create the lived experience of functioning and disability . Although not classified in icf, contextual factors also include personal factors such as sex, age, coping styles, social background, education, and overall behavior patterns that may influence how disability is experienced by the individual . The terms functioning and disability in the icf are the general or umbrella terms for, respectively, the positive and negatives aspects of the interaction between an individual (with a health condition) and that individual's contextual factors (environmental and personal factors). In the icf, health condition is the umbrella term for disease (acute or chronic), disorder, injury or trauma . A health condition may also include other circumstances such as pregnancy, aging, stress, congenital anomaly, or genetic predisposition . The icf interactive model identifies three levels of human functioning: functioning at the level of body or body part, the whole person, and the whole person in their complete environment . These levels in turn define three aspects of functioning: body functions and structures, activities, and participation . Disability similarly denotes a decrement in functioning at one or more of these levels that is, an impairment, activity limitation or participation restrictions . Table 2 shows the complete list of all of the chapters found in the three classifications included in icf . Under each of these chapters are second, third, and in some instances, fourth levels of categories, arranged in a hierarchical, tree - branch - stem - leaf, arrangement . This structure makes it possible for icf to be used as a classification tool for systematically describing situations of human functioning and problems with functioning . This complex information is organized by icf by means of a hierarchical coding system, thereby creating a common international language for functioning and disability . Icf organizes information by means of several classifications distributed into two parts: (1) a component of functioning and disability that includes the component of the body with the body function and body structure classifications, and the component of activities and participation that includes all domains denoting aspects of functioning from an individual and social perspective organized into a single classification, and (2) a component of contextual factors that has a list of environmental factors organized from the individual's most immediate to the wider environment . The classifications in the first part identify all of the domains of functioning from basic physiological functions and body structures, to simple and complex actions, tasks, social performances and relationships . The environmental factors list provides a tool for identifying those features of a person's physical, human - built, social and attitudinal environment that, in interaction with the domains of functioning, constitute the complete lived experience of human functioning and disability . Within the contextual factors part, besides the environmental factors, the icf recognizes the existence of personal factors as another component, but provides no classification of these . Domains are a practical, meaningful set of related physiological functions, anatomical structures, actions, tasks, or areas of life . Domains make up the different chapters and blocks within each component (world health organization, 2001). In order for these domains to capture descriptive information about functioning and disability in particular cases, they must be used in conjunction with qualifiers that record the presence and severity of a problem or decrement in functioning at the body, person, and social levels . For the classifications of body function and structure, the primary qualifier indicates the presence of an impairment and, on a five - point scale, the degree of the impairment of function or structure (no impairment, mild, moderate, severe, and complete). In the case of the activity and participation list of domains, two essential qualifiers are provided to capture the full range of relevant information about disability . The performance qualifier is used to describe what an individual does in their current or actual environment, including whatever assistive devices or other accommodations the person may use to perform actions or tasks and whatever barriers and hindrances exist in the person's actual environment . Because the current environment always incorporates the overall social context, performance might be understood as involvement in the lived experience of disability . The capacity qualifier describes an individual's inherent ability to execute a task or an action . Operationally, this qualifier identifies the highest probable level of functioning of a person in a given functional domain at a given moment without any specific assistance . For measurement purposes, this level of capacity presumes a standardized assessment environment, namely one that reveals the inherent capacity of a person in a specific functional domain without any particular enhancements . The environmental factors list can be used to describe such a standard assessment environment in order to ensure that results across different studies can be compared by holding this environment constant . Intuitively, the performance qualifier captures what people actually do in their lives, whereas the capacity qualifier identifies the person's inherent capacity without explicit environmental facilitation (or hindrance). Who is developing a standard application guide that will operationalize the constructs of capacity and performance with respect to individual items that form the classification . Table 3 shows how data can be organized to reflect the role of these two qualifiers used for the domains of the activity and participation classification . As a general matter of describing functioning and disability phenomena fully and accurately, the performance / capacity having access to both performance and capacity data enables icf users to determine the gap between capacity and performance . If capacity is less than performance, then the person's actual or current environment has enabled him or her to perform better than what data about their capacity would predict: the environment has facilitated performance . On the other hand, if capacity is greater than performance, then some aspect of the environment is acting as a barrier to a level of performance that is feasible in a more suitable environment . Icf thus makes it possible to measure the effect of a person's environment on their decrement in functioning, given their health condition . The environmental factors classification can be used to identify specific features of the person's actual environment that are barriers or facilitators in general for the person or with specific regard to each item of the person's body functions, body structures or activities and participation that have been described . It can also be used, as previously stated, to describe specific testing environments where capacity has been measured . For its use as a classification of functional status relevant for health administrative records, icf provides a complete classification of both body and person level domains of functioning . Given that it has been designed for a multiplicity of uses and users, there is far more in icf than could ever be plausibly integrated into a viable coding system for health records, although it remains the ultimate lexicon to which any coder, for clinical or research purposes, could turn . Clearly, for implementation purposes in this area, a simplified checklist of items is needed . Such a checklist was produced and used during the beta 1 and 2 field - testing phase in the revision process (world health organization, 2001). This checklist, which takes less than 30 minutes to complete, is currently being extensively tested in clinical studies in different disorders in order to study its feasibility, reliability, and concurrent validity with existing assessment instruments as part of a larger project to define core sets of items that may be used in rehabilitation settings for specific conditions and across several disorders (stucki et al ., 2002). The core sets of items with their corresponding scales could also be then converted into even shorter assessment instruments . The challenge for incorporating the icf into clinical and administrative records beyond a lexicon and framework lies in identifying this parsimonious set of domains or items that captures decrements in functioning across different health conditions and a smaller subset of domains or items that uniquely describe the decrements of functioning that typify a given health condition . In addition, the mapping of instruments (that measure functioning and disability that are already in use) onto icf categories will allow a ready crosswalk between measurements already being made at points of encounter to a common framework (cieza et al ., 2002). The use of the icf in larger population based surveys will also provide data on norms and distributions of health, functioning and disability that will enable the setting of appropriate thresholds for a multitude of purposes . Table 4 maps the domains of the icf that have been included in different waves of the world health survey that ought to be included as a minimum or ideal set for information systems . These domains are also included on the icf checklist, which is designed to be a clinical tool . Primary data collection strategies with regard to functional status, in a manner that is truly comparable, are in their infancy especially for international use and for use across population groups . Further tools need to be developed, and standards and procedures established, so that these data become meaningful and usable . As a final issue, it must be mentioned that the icf has been conceived as a dynamic classification that will not only serve multiple users requiring different levels of detail, but also will continue to evolve with advancements in science . The classification is flexible in its structure such that it can be expanded in the level of detail (for example, the fourth level) for specific uses, or new codes added where gaps have been left in the numbering system . A set of operational rules will specify the procedure for this evidence - based expansion, adaptation, or revision of the classification . A common language for describing fsi is the key to ensuring comparability of data from a myriad of sources as well as in providing users with a tool for precise and accurate communication with each other . The recognition that a description of health and health - related outcomes must go beyond a narrow view of health restricted to the absence of disease, as well as that the definition of disability must move beyond the narrow impairment - based view that has been traditionally adopted to define a minority population, will go a long way in bridging the gap between health and disability data . It will also fill the void in existing health outcomes data while measuring the impact of interventions and monitoring them over time . Health records must include functioning information in order to ensure a complete description of health states . The icf is the common language and framework that users will employ from now on . In the same way that all languages grow, evolve, and flourish over time and are adapted and modified to express new ideas, the icf will have a multitude of applications where it will be creatively used such that it continues to be a living classification . As with all new languages, it will be important to develop tools to learn this new language . Toward this end, who is developing standardized application manuals and web - based learning courses that will use state - of - the art pedagogic methodology to assist end users . Its usefulness in describing functional health status information will be one of the measures of its success.

The centers for disease control and prevention (cdc) and united states preventive services task force (uspstf) recommend universal hiv screening for all pregnant women entering prenatal care [1, 2]. This screening enables hiv - infected women and their infants to benefit from appropriate and timely interventions such as antiretroviral medications . When the recommended antiretroviral and obstetric interventions are used, a woman who knows of her hiv infection early in pregnancy now has a less than 2% chance of delivering an hiv - infected infant . Without intervention, this risk is approximately 25% in the united states [36]. Testing for hiv began in 1985 with the introduction of the enzyme immunoassay (eia). In order to account for false positive results using screening tests in a low - prevalence population, confirmatory testing has been implemented using a western blot or immunofluorescence assay . In a low - prevalence population, the false positive rate using the eia is increased compared to a high - prevalence population, and the positive predictive value of any test will always depend on the prevalence of the condition in the population tested . In testing performed by the cdc, the eia positive predictive value ranges from 71 to 83% in populations with hiv prevalence from 0.5 to 1% . Some investigators believe that the presence of alloantibodies account for the increased false positive rate associated with pregnancy, transfusions, transplantation, and autoimmune diseases . However, risk factors specific to pregnancy that account for this are poorly understood . Conversely, a recent large retrospective study found that the false positive hiv eia rate was lower in pregnant women compared to nonpregnant individuals (0.14% versus 0.21%). Our objective was to determine if any maternal characteristics correlated with false positive hiv eia testing at delivery . This was a review of all women who delivered at parkland memorial hospital in dallas, tex, from october 1, 2005 through september 30, 2008 . All women routinely received serum hiv testing at their initial prenatal visit and at time of presentation to labor and delivery for delivery via the opt - out approach with the abbott commander hiv ab hiv-1/hiv-2 (rdna) eia (abbott laboratories, abbott park, ill). Hiv test results performed at the time of delivery were analyzed in this study . A woman was considered hiv negative if eia testing was negative . Positive test results were confirmed with the fluorognost immunofluorescent assay (ifa) hiv-1 (sanochemia corp, stamford, conn, usa). Women were considered to be falsely positive if eia results were positive and the ifa was negative . Women delivered in this time period were identified through the obstetric operations database and linked to the pathology database for hiv, hepatitis b surface antigen (hbsag), and rapid plasma reagin (rpr) results . Maternal characteristics, including race, parity, age, singleton versus multiple gestation, and a diagnoses of diabetes or hypertension were obtained using the obstetrics operations database . Laboratory results drawn 28 days prior to delivery through seven days after delivery were included . The study was approved by the institutional review board at the university of texas southwestern medical center . Categorical data were reported as frequencies, and statistical significance was determined using analysis . Statistical analyses were performed using sas, version 9.2 (sas institute, cary, nc). A total of 47,794 women were identified who delivered during the study time frame . Compared to hiv negative patients, false positive patients were more likely to be nulliparous (43% versus 31%, p <0.001) and younger (mean age 23.9 5.7 versus 26.2 5.9, p <0.001). Hiv positive patients were significantly older than false positive patients (27.4 versus 23.9, p <0.001) and hiv negative patients (27.4 versus 26.2, p = 0.012). Of the 47,794 women, 47,391 (99%) were hiv negative, 145 (0.3%) had a false positive test, 172 (0.4%) were true positives, and 86 (0.2%) tested equivocal or were missing hiv results . The specificity of the hiv eia test was 99.7% with a positive predictive value of 54.3% . The sensitivity of the eia test was presumed to be near 100%, as the false negative rate using the eia has been previously demonstrated to be negligible in studies performed by the manufacturer . Table 2 shows the prevalence of diabetes, hypertension, hepatitis b, and results of syphilis testing in the study population . Hiv positive women were more likely to test positive for hepatitis b surface antigen (1% versus 0%, p = 0.002) and rpr (2% versus 0%, p = 0.02). There was no significant difference in the prevalence of diabetes or hypertension between the three groups . There was also no significant difference in the rate of hbsag and rpr positivity between the hiv negative and false positive groups . When evaluating the interaction between nulliparity and age, there is a significant correlation between the two variables; that is, parous patients are likely to be older and, therefore, nulliparity and age are not independent predictors of hiv false positivity . However, when the hiv testing groups (positive, false positive, negative) are stratified by parity, the comparison of age across the three groups remains significant only in nulliparous women (table 3, p = 0.0003). The interaction between parity and age means that the difference in age between nulliparous and parous women is different depending on the hiv status . For example, in the hiv positive group the nulliparous and parous women are closer in age than in the false positive group . This was the first population - based study to evaluate risk factors for hiv false positivity in pregnant women presenting for delivery at a large urban institution . We found that younger and nulliparous women were more likely to have false positive testing using the hiv eia . The observed positive predictive value (ppv) of 54.3% was lower than the previously reported by the cdc (ppv 7183%) in a nonpregnant population, suggesting that pregnancy may be associated with a higher rate of false positivity . The low positive predictive value of the hiv eia in our study may have been impacted by the relatively low hiv prevalence (0.4%) in our population . This information could be useful for counseling women who test positive for hiv at delivery and emphasizes the limitations of eia testing if used as a rapid test to determine the need for intrapartum and neonatal antiretroviral prophylaxis . The false positive rates of currently available rapid hiv tests have been reported to be much lower than has been found with eia testing in this study . Evaluated the hiv false positive rate in over 900 pregnant women, most of whom were hispanic, using both the eia and a rapid point - of - care (poct) test, oraquick . They found that while there were no false positive tests with the oraquick, there were seven false positives using the eia (ppv 100% versus 35.7%). In the mother - infant rapid intervention at delivery (miriad) study, jamieson et al . Found that the ppv of the oraquick test was 90% while the ppv of the eia was 74% [12, 13]. Current recommendations by the cdc and the american college of obstetricians and gynecologists include rapid hiv screening for women presenting to labor and delivery with undocumented hiv status and for repeat hiv testing in the third trimester for women at high risk or who live in areas with high hiv prevalence [7, 14, 15]. A woman testing preliminarily positive for hiv in labor should be counseled that she may have hiv infection and that her neonate may be exposed, and immediate antiretroviral prophylaxis should be recommended without waiting for confirmatory test results . The results of our study may aid in counseling women if they test preliminarily positive for hiv using the eia, while awaiting confirmatory testing results . It remains to be determined whether the same risk factors for false positive hiv eia testing apply to the poct tests used in a real life setting . Our study found that the positive predictive value of eia testing was only 54.3% and that younger nulliparous women were more likely to test falsely positive . Importantly, our data may represent the real world performance of the eia testing in a large obstetric population - based setting . Investigators also have noted a significant relationship between influenza vaccination and false positive screening for hiv antibodies [1618]. A potential reason for this cross reactivity is that there are similarities in homology between the transmembrane domains of the influenza envelope protein hemagglutinin and the hiv-1 envelope proteins . In our population, there were no significant differences in age or parity and influenza vaccination acceptance rates (unpublished data). Women in our study were from a single institution, with a predominantly hispanic background, and therefore the results may not be applicable to all populations . Reasons for false positive hiv serology may vary depending on the geographical region . While we did find a significant association between young age, nulliparity, and hiv false positive testing, our study does not have the capability to identify a causal relationship or explain why this association may exist . In addition, our study cannot address if these same risk factors for false positive testing apply to all other hiv tests . Further studies are needed to confirm our findings, elucidate the biological mechanisms for increased hiv eia false positivity in young, nulliparous women, and compare this conventional testing method with contemporary rapid screening assays, including poct.

Childhood obesity is a serious and growing public health problem that has arisen over the past three decades . The increasing occurrence of disorders such as type 2 diabetes during childhood is believed to be a consequence of this obesity epidemic . In addition to several behavioral and dietary risk factors, genetic predisposition is an important factor in the pathogenesis of childhood obesity . It is estimated that 4070% of adiposity variance can be explained by direct or indirect genetic factors . Growth arrest - specific 6 (gas6), cloned in 1988 and characterized in 1993, is a secreted vitamin k - dependent protein present in the human circulatory system [4, 5]. Initially, gas6 was shown to be upregulated in growth - arrested fibroblasts, suggesting that it plays a protective role in certain cellular stresses such as during apoptosis . Gas6 expression is widespread in many tissues, including immune cells, endothelial cells, vascular smooth muscle cells, and adipocytes [79]. The protein is also a ligand for the tam (tyro-3/axl / mer) family tyrosine kinase receptor . The gas6/tam system has been implicated in cell survival and proliferation, cell adhesion and migration, hemostasis, and inflammatory cytokine release [4, 10]. Recently, the gas6/tam pathway was found to be involved in mediating adipocyte survival and proliferation in vitro [11, 12]. Experiments with mice fed a high - fat diet indicated that overexpression of gas6 might enhance body - fat accumulation, but blocking gas6 signaling using an axl antagonist could reduce body - fat mass and body weight . Interestingly, transgenic animals that ectopically express the axl tyrosine kinase receptor also develop progressive obesity with elevated circulating proinflammatory cytokines and severe systemic insulin resistance . This protein - array study also revealed higher levels of axl mrna in subcutaneous adipose tissue of obese humans than their lean control counterparts had . This indicates that the axl receptor may be involved in the development of human obesity . In addition, some studies in transgenic mice indicate that gas6/axl signaling might recruit macrophages and other immune cells into the adipose tissue resulting in the production and secretion of proinflammatory mediators . This suggests that the gas6/axl signaling might play a role in the pathogenesis of obesity - associated systemic inflammation [8, 16, 17]. Recent studies have indicated that systemic inflammation, a hallmark of childhood and adult obesity, is a pivotal mechanism linking obesity to insulin resistance and type 2 diabetes [1821]. Although gas6 gene polymorphisms are reported to be associated with stroke, acute coronary syndrome, and type 2 diabetes [2224], to our knowledge, both gas6 and axl gene polymorphisms associated with childhood obesity have not yet been identified . In order to address this issue, we conducted a community - based study to determine whether common variations in the gas6 and axl genes correlate with adiposity, systemic inflammation, insulin resistance among adolescents . The taipei children heart study - iii was an epidemiological survey that investigated obesity and cardiovascular disease risk factors among adolescents in taipei city during 2006 . The sampling method and results briefly, the survey included junior high school students in taipei city to collect a representative distribution of demographic, lifestyle, and biochemical characteristics to measure their risk for cardiovascular disease . Those with autoimmune diseases, cancers, or active infection and those taking medications known to interfere with insulin or glucose metabolism were excluded . Excluding any missing data, 727 adolescents (358 boys and 369 girls) the institutional review board of the tri - service general hospital approved these studies and obtained informed consent from both parents and adolescents . All the participants completed a structured questionnaire detailing their gender, age, puberty development, and lifestyle characteristics (including cigarette smoking, alcohol consumption, and physical activity). Based on their responses, the subjects were divided into young adolescents with history of smoking, those without, and those who currently smoke . Physical activity was divided into 5 levels based on amount of exercise per week: less than 1 h, 13 h, 35 h, 57 h, and over 7 h. survey questions concerning puberty onset included the development of the penis / testis and pubic hair for boys and development of breasts and pubic hair for girls . Body weight was measured of barefoot students wearing light indoor clothing and was rounded to the nearest 0.1 kg . Waist circumference (wc) was measured at the midway point between the inferior margin of the last rib and the crest of the ilium in a horizontal plane and was recorded to the nearest 0.1 cm . Body mass index (bmi) was calculated as weight in kilograms divided by the square of height in meters . To reduce extraneous variation between subjects, we collected blood samples from the students after 12 h fasting and who had consumed their usual diet for the previous 3 days . Children who had recently attended a holiday or family celebration were contacted for a blood sample several weeks after the event . Plasma glucose concentrations were determined by the glucose oxidase method by using the beckman glucose analyzer ii (beckman instruments, fullerton, ca). The intra- and interassay coefficients of variation (cvs) for glucose were 0.6% and 1.5%, respectively . Plasma insulin was measured using a commercial immunoradiometric kit (biosource europe, nivelles, belgium). Serum levels for high - sensitivity c - reactive protein (hscrp) were measured using the tina - quant (latex) high - sensitivity assay (roche, mannheim, germany). Serum il-6 concentrations were determined using a human high - sensitivity enzyme - linked immunosorbent assay (elisa) (innotest, besancon, france). Serum tnf- was measured with the biotrak high - sensitivity human elisa kit from amersham biosciences (buckinghamshire, uk). Insulin resistance was assessed using the homeostasis model assessment (homa), in which the homa of insulin resistance (homa - ir) = fasting insulin (u / ml) fasting glucose (mmol / l)/22.5 . Gas6 protein concentration was measured using a sandwich elisa and a polyclonal mouse anti - human gas6 antibody (r&d systems, lille, france) as a catcher and a biotinylated goat antiserum as a detector (r&d systems), using previously described methods . The technique has been validated by food and drug administration guidelines in a previous study (intra- and interassay cvs of 6.5% and 8.5%, resp . ; mean recovery on 10 patients of 97%; lower limit of quantification 0.26 ng / ml). Dna was isolated from buffy coats using the qiaamp dna blood kit and following the manufacturer's instruction (qiagen, valencia, ca, usa). The qualities of isolated genomic dnas were quantified using agarose gel electrophoresis, and the quantities were determined using a spectrophotometer . Snps rs8191973 and rs8191974 in gas6, as well as rs4802113 and rs2304232 in axl, were genotyped using a taqman assay with allele - specific probes on the abi prism 7900ht sequence detection system (applied biosystems, foster city, ca, usa) according to the standardized laboratory protocols . Descriptive results of continuous variables were expressed as means sd . Prior to statistical analysis, the normal distribution and homogeneity of the variables were evaluated using the levene test for quality of variance, and the variables were then given a base logarithmic transformation if necessary . The parameters homa - ir, triglycerides, hscrp, il-6, and tnf- were analyzed and tested for significance using a log scale . The studied adolescents were categorized into subgroups based on their gas6 rs8191973 genotype (cc, cg, and gg), gas6 rs8191974 genotype (gg, ga, and aa), axl rs4802113 genotype (cc, ct, and tt), and axl rs2304232 (aa, ag, and gg) with gender specification . The differences between anthropometric and biochemistry data across genotypes were analyzed using a general linear model after adjusting for age, tanner stages, smoking status, drinking status, and physical activity . Chi - square tests were used to determine the genotype distributions for the hardy - weinberg equilibrium and to compare the proportions of abnormal anthropometric and biochemistry variables across genotypes . We tested different genetic inheritance models, and a recessive model was applied in the final analyses for gas6 and axl . To determine whether the gas6 and axl snps are predictors of obesity and obesity - associated complications, logistic regression analysis was used to calculate the odds ratio (or) and 95% confidence interval (ci) for each genotype and combined genotypes . A two - sided p - value of <0.05 was considered statistically significant . All statistical analyses were performed using pasw statistics 18.0 software (spss inc ., chicago, il, usa). In total, 727 adolescents (358 boys and 369 girls) were included in this study . The mean age of all adolescents in this study was 13.3 years (range, 1215) and was similar between boys and girls . In general, boys had higher bmi (22.3 4.0 versus 21.2 3.3 kg / m), wc (80.0 10.1 versus 75.1 8.1 cm), hscrp (0.9 1.3 versus 0.6 0.9 mg / l), and glucose levels (93.8 6.3 versus 91.5 6.5 mg / dl) than the girls (all p <0.001). However, girls had higher tanner stages (3.2 0.5 versus 3.0 0.4) than the boys (p <0.001). There was no statistically significant difference in the ages, tnf-, il-6, and insulin levels, and homa - ir between the boys and girls . The allele frequency for the least frequent allele in boys was 12.6, 22.1, 41.9, and 29.7%, and 13.4, 19.6, 32.7, and 33.5% in girls for the gas6 rs8191973, gas6 rs8191974, axl rs4802113, and axl rs2304232 polymorphisms, respectively . There was no significant difference in allele or genotype distribution between boys and girls at the 4 polymorphisms . No statistically significant association between anthropometric characteristics, biochemistry data, and the gas6 rs8191973 genotypes was observed in the boys and girls (table 1). However, there were significantly different hscrp levels between gg, ga, and aa genotypes of gas6 rs8191974 in boys, regardless of their age, tanner stages, smoking status, drinking status, or physical activity (table 2). Moreover, boys with the gg genotype of gas6 rs8191974 had significantly higher bmi, wc, and hscrp levels than those carrying the a allele . The gas6 rs819174 genotypes were not significantly associated with any anthropometric characteristics and biochemistry in girls . The p - values of all comparisons between anthropometric and biochemistry data across gas6 genotypes were presented in supplemental tables 1 and 2 available online at http://dx.doi.org/10.1155/2014/674069 . In addition, the association between circulating gas6 protein levels and gas6 polymorphisms was investigated . We found that the gas6 rs8191973 or rs8191974 genotypes were not significantly associated with circulating gas6 protein levels in both the sexes . There were significantly different hscrp levels between tt, tc, and cc genotypes of axl rs4802113 in boys, independent of their age, tanner stages, smoking status, drinking status, or physical activity (table 3). In addition, boys with the gg genotype of axl rs2304232 had significantly higher il-6 and insulin levels and increased homa - ir than those carrying the a allele (table 4). However, in girls, axl rs4802113 or rs2304232 polymorphisms were not significantly associated with any anthropometric characteristics or biochemistry (tables 3 and 4). The p - values of all comparisons between anthropometric and biochemistry data across axl genotypes were presented in supplemental tables 3 and 4 . Boys with the gg genotype of gas6 rs8191973 were 1.87-fold more likely to have higher hscrp levels than the c allele carriers . Even after adjusting for age, tanner stage, smoking status, drinking status, and physical activity, a significant relationship between the gg genotype of gas6 rs8191973 and higher hscrp levels was still observed in boys (table 5). Moreover, boys with the gg genotype of gas6 rs8191974 exhibited a 1.40-fold greater risk for developing high bmi, a 1.58-fold greater risk for developing high wc, and a 2.68-fold greater risk to have higher il-6 levels than the a allele carriers . Even after adjusting for all possible confounding factors including age, tanner stage, smoking / drinking status, and physical activity, the relationship between the gg genotype of gas6 rs8191974, higher bmi / wc, and higher il-6 levels still remained significant in boys . However, the axl rs4802113 or rs2304232 polymorphisms showed no significant association with abnormal adiposity, inflammatory markers, and homa - ir in boys or girls (see supplemental table 5). Logistic regression analyses were applied to evaluate whether the combination of the gas6 rs8191974 and rs8191973 polymorphisms is a stronger risk factor for high adiposity, inflammatory markers levels, and homa - ir than when alone . The combined effects of the 2 gas6 gene polymorphisms in the risk of high bmi, wc, il-6, and hscrp levels are shown in figure 1 . After adjusting for the relevant confounding factors, we still observed that boys with the gg genotype of gas6 rs8191973 and the gg genotype of gas6 rs8191974 exhibited a 47-fold higher risk of high bmi, wc, il-6, and hscrp levels than the individuals with both the c allele of the gas6 rs8191973 and the a allele of the gas6 rs8191974 did (or = 4.92, 95% ci: 1.0823.6, p = 0.018; or = 4.18, 95% ci: 1.0522.5, p = 0.016; or = 4.08, 95% ci: 1.0628.56, p = 0.015; or = 7.22, 95% ci: 1.4635.72, p = 0.010, resp . ). However, for girls, there was no statistically significant association between the combination of the gas6 rs8191974 and rs8191973 polymorphisms and abnormal variables . In addition, we evaluated the combined effect of the gas6 rs8191973 or rs8191974 marker with axl gene polymorphisms and its association with risk of high adiposity, inflammatory marker, and homa - ir . However, combinations of gas6 markers with axl gene polymorphisms were not found to be significantly associated with any abnormal variables in both boys and girls (data not shown). In this study, a strong association between gas6 and axl polymorphisms with body adiposity, systemic inflammation, and insulin resistance was identified among boys . The risk of possessing high adiposity and inflammatory markers levels was higher in boys carrying the gg genotype with gas6 rs8191973 or rs8191974 than the noncarriers . Moreover, the combination of both gas6 polymorphisms had an additive effect on the development of obesity and obesity - associated inflammation in boys . These data strongly suggest that gas6 and axl genes play a role in the pathogenesis of childhood obesity and its associated complications . Gas6 was originally identified as a gene that is expressed in fibroblasts and increases with serum starvation and contact inhibition; gas6 is also a potential growth factor for fibroblasts . Maquoi and colleagues demonstrated that when fed with a high - fat diet, gas6-deficient mice had significantly less fat than their wild - type counterparts . The authors also reported the expression of gas6 and its 3 receptors (tyro-3, axl, mer) in murine adipose tissues, thus suggesting that gas6 may act in an autocrine and/or paracrine manner to promote murine adipose tissue development . Previous experiments in transgenic mice demonstrate that gas6 might also induce obesity - associated inflammation via recruiting immune cells into the adipose tissue to producing and secreting proinflammatory cytokines [8, 16, 17]. Our recent clinical study found that circulating gas6 protein levels are associated with adiposity and inflammatory markers in overweight / obese adolescents . In this study, gas6 is further implicated as a candidate susceptibility gene for obesity and systemic inflammation . However, the association between gas6 genotypes and circulating gas6 protein levels was not observed among adolescents . We hypothesize that gas6 polymorphisms could affect the biology of the gas6 protein itself rather than its transcription or process rate, thus influencing adiposity regulation and systemic inflammation . To validate this, recent studies demonstrated that gas6/tam signaling is involved in releasing inflammatory cytokines (such as il-6 and hscrp) in diverse human diseases [23, 31, 32]. In addition, the gas6/tam signaling is also known to be involved in several inflammation - related systems, including maturation of immune cells, endothelial activation, and immunoregulation . Our present study found that the gg genotype of gas6 rs8191973 and the gg genotype of gas6 rs8191974 are strongly associated with higher circulating il-6 and hscrp levels in boys . Therefore, the gas6 polymorphisms presumably influence gas6/tam signaling and could further activate inflammatory reactions and result in releasing circulating il-6 and hscrp . However, the comprehensive effects of the gas6 polymorphisms in regulation of inflammatory cytokines still remain to be determined by more researches . Interestingly, a previous study found that the a allele or the aa genotype of gas6 rs8191974 is associated with decreased risk of stroke . Moreover, the a allele and the aa genotype are also thought to be related to a lower risk of developing acute coronary syndrome or type 2 diabetes, suggesting that this genotype exhibits protective activities against developing acute coronary syndrome and type 2 diabetes [23, 24]. We also observed similar results in those with the a allele or aa genotype of gas6 rs8191974 . These subjects exhibited a lower risk for developing obesity and systemic inflammation than those with the gg genotypes . Together, these findings suggest that the gas6 rs8191974 polymorphisms play an important role in the development of obesity and obesity - associated complications (e.g., type 2 diabetes, cerebrovascular, and cardiovascular diseases). The protective role of the aa genotype of gas6 rs8191974 against the developing childhood obesity and obesity - associated complications requires further study . The axl protein is a membrane - bound receptor that belongs to the tam family of receptor tyrosine kinases . Axl exhibits the highest affinity for gas6 as compared to the other members of the tam family, whereas protein s predominantly binds mer and tyro-3 . Gas6/axl signaling has been shown to be involved in the pathogenesis of obesity and systemic inflammation [1315]. However, our study demonstrates that axl polymorphisms are associated with systemic inflammation rather than childhood obesity . Moreover, the combination of gas6 and axl gene polymorphisms is not significantly associated with any variables in adiposity among adolescents . Our findings indicated that axl gene polymorphisms might not play a significant role in childhood obesity . Recently, scroyen and colleagues have published similar findings indicating that deficiency in a single axl receptor did not significantly affect adipogenesis or adipose tissue development in mice . This is because an axl deficiency can be partially compensated by other tam family members (tyro-3 and mer) via gas6 interaction . Further studies are needed to investigate the effect of tyro-3 and mer receptors on the development of childhood obesity . In addition, our present study also indicates that gender disparity exists regarding the effects of the gas6 polymorphisms on anthropometric characteristics and inflammatory markers . We found no significant difference in genotype frequencies between boys and girls; however, the effects of the gas6 polymorphisms, individually or combined, only manifest in boys . The gas6 gene contains an estrogen response element in its promoter and is upregulated by estrogen via an activated estrogen receptor in mammary epithelial cells . Therefore, we hypothesized that the gender - specific effect of the gas6 polymorphisms on childhood obesity might be due to a disparity in sex hormone distributions . This has been previously reported to be associated with gas6 expression and body composition [40, 41]. Despite these results, our study does have certain limitations . First, this was a cross - sectional study, as such we might not be able to assess gas6 polymorphisms on weight dynamics and the development of obesity - associated complications throughout life . Second, because of the limitations of our questionnaire, we were not able to comprehensively estimate every adolescent's daily intake . The impact of dietary energy intake on genetic susceptibility also requires further investigation to better understand any confounding effect . In conclusion, we indicate an association between the gas6 and axl polymorphisms with adiposity, circulating inflammatory markers, and insulin resistance of adolescents, especially in boys . Moreover, the gg genotype of gas6 rs8191973 or rs8191974 strongly correlates with susceptibility to develop obesity and systemic inflammation in boys . Nonetheless, these results together with those from studies in cellular and animal models encourage the study of the gas6/tam system in childhood obesity and its potential complications and further support the hypothesis that modulation of gas6 activity may indeed provide an important intervention point for future therapies.

Infertility is one of the crucial and critical events in sex life which engages 1015% of couples all over the world, and as one of the most distressful life experiences, exposes couples to social and psychological problems . Couples, who are faced with this critical situation, are prone to depression, anxiety, loss of self - esteem and dissatisfaction with their sex life more than others . However, the intensity of psychological problems resulting from cultural - social circumstances varies in different societies, to the point that the frequency of anxiety in infertile couples has been reported in a wide range from 48% to 96% . Although, the advances in assisted reproductive techniques have opened new doors for infertile couples, but studies have shown that these treatments are accompanied with stress, anxiety and depression . But the intensity of these disorders could be defined by the cause of the infertility . Evaluating the related factors of depression and anxiety among iranian infertile couples showed that women who were infertile themselves showed higher levels of anxiety and depression than women with male factor infertility . However, in societies that define women as the main source of fertility, starting assisted reproductive treatments with limited successful outcomes could be harmful to these women's mental health . Utilization of assisted reproductive techniques requires processes such as daily injection to stimulate ovulation, vaginal ultrasound and painful processes such as oocyte aspiration which are all too expensive and alongside with the fear of failure could become a harmful condition . Although, fertile women, who start assisted reproductive treatments due to their husband's infertility have natural biological conditions to get pregnant, but they have to tolerate these critical processes due to male factor infertility . Also, women are under more cultural and social pressures . These conditions could affect the vulnerability of fertile women during assisted reproductive treatment; therefore the aim of this study was to evaluate the effect of assisted reproductive treatments on fertile women's mental health . Thus, the prospective study conducted on 70 fertile women who referred to receive assisted reproduction treatments (in vitro fertilization / intracytoplasmic sperm injection) due to male infertility . Subjects had referred to the fertility and infertility center of esfahan from september 2013 to march 2014 . The number of samples was calculated based on 80% test power and 95% confidence level . Inclusion criteria were primary infertility, not having any corporal and mental illnesses, not being a drug addict according to individual's report, not having a history of mental illnesses, and not experiencing severe distress over 1-month prior to the treatment based on holmes - rahe scale . Exclusion criteria included experiencing distressful conditions during the study, discontinuation of the treatment and the cancellation of egg harvesting . The study was approved by the medical ethics committee of the isfahan university of medical sciences . Mental health evaluation tool was the valid 28-questioned general health questionnaire that measures mental health on likert scale (03) in hypochondriasis, anxiety, social impairment, and depression dimensions . In this method gaining the score of 5 in each subscale and 23 in all dimensions together was defined as having a mental disorder . Sampling was conducted through simple sampling; that is, all persons, who referred to fertility and infertility center of esfahan for treatment and to start the process protocol due to male factor infertility were interviewed . Then by referring to their medical records and conducting holmes - rahe scale, their eligibility for the study was assessed . After being considered eligible for entering the study, their demographical features including age, educational level of couples, economic condition, and the duration of their infertility were recorded . Ovulation was stimulated through the long protocol in all the participants . During the treatment period, ovarian reaction conditions and number of injections employed for ovarian stimulation were followed - up . 3 h after the pickup process, and under appropriate circumstances, the questionnaire for general health was completed again . If the circumstances were inappropriate filling of the questionnaire was delayed for 2 h. research data were analyzed using spss software version 16 [spss inc . : chicago, il, usa], statistical paired t - tests and multi - variable linear regression tests . Seven qualified subjects quit the study because of the unwillingness to continue (three persons) and ovarian hyper stimulation (four persons). Demographic and clinical data results showed no significant difference in the mean score of different dimension of mental health before ovulation induction and after oocyte harvesting [table 2]. Also, the rate of psychological disorder in the anxiety dimension showed no significant different before the induction (71.4%) and after the pickup (66.7%) (p = 0.19). But the level of depression after pickup (31.7%) was significantly lower than before the induction (39.7%) (p = 0.007). Comparison of mental health dimensions b and a results showed no significant difference in the dimension of hypochondriasis before the induction (61.9%) and after the harvesting (66.7%) (p = 0.07). Also, the level of social impairment showed no significant difference before the induction (84.1%) and after the pickup (87.3%) (p = 0.08). The results of multi - variable linear regression to evaluate the relation between underlying variables and mental health condition before starting the process with the level of depression and anxiety after the harvesting are shown in table 3 . The relation between mental health dimensions and background variables after ovulation stimulation the level of depression after egg harvesting was related to physical condition, depression and social impairment, independent from underlying variables . Also, the level of depression after egg harvesting was related to the economic condition, it also showed a significant relation with social impairment and hypochondriasis [table 3]. The present study aimed to assess the level of mental health in fertile women undergoing assisted reproductive treatments as a result of male factor infertility, at the beginning of ovulation stimulation process, and the effect of this process on their mental health . Results showed that these women do not start assisted reproduction treatment under appropriate mental health conditions . But the level of depression and anxiety disorders at the beginning of ovulation stimulation was higher in the present research than other reports . This would indicate that the process of treatment in fertile women is also a critical one and might affect their mental health . In a pursuit, to achieve a conceptual model for the effect of ovulation induction on women's health, its negative effects on women's functioning and health have been shown . A qualitative study showed that iranian infertile couples show emotional reactions such as fear, concern, anxiety and depression as a result of fear of the failure of treatment during fertility treatment . However, these mental disorders may be harmful even for women with appropriate fertility potentials . Also being angry with their husbands due to their infertility could be another explanation for women's mental reactions . Positive correlation between the level of depression and anxiety and other dimensions of experiences during infertility treatment might be accompanied with social impairment, thus affecting the levels of depression and anxiety . Efforts to conceal infertility and efforts to get treatment as a result of concerns about being labeled as infertile, are common among iranian couples . This, by itself, could increase the level of anxiety among women when treatment starts . Another reason for the high level of depression and anxiety among women at the beginning of the ovulation stimulation might be the negative impact of gonadotropin - releasing hormone (gnrh) agonists on the mental health . Although, this research did not evaluate mental health before and after the beginning of gnrh agonists, but this might explain women's psychological disorders before ovulation stimulation regarding high frequency of mental disorders at the time of ovulation stimulation . With regards to the negative impact of anxiety and depression during the treatment on the result of assisted reproduction treatment, it is necessary that this group of women would be examined before the treatment process, since they are exposed to risks of mental health disorders, and be treated in case of high levels of depression and anxiety . Following the assessment of the main hypothesis of the research, that is the effect of ovulation stimulation on women's mental health, study results showed that ovulation stimulation does not affect mental health; the level of depression and anxiety, and the measure of psychological disorders in these two - dimensions did not show a significant difference before the ovulation and after the harvesting process . On the contrary, the depression level was decreased after the harvesting process . The negative impact of distress of infertility treatment on the level of depression has already been reported . The cessation of treatment distress with the conclusion of ovulation stimulation and harvesting processes might be a reason for the decrease of depression level in subjects under study . Furthermore, results showed that fertile women's level of depression and anxiety does not depend on the reaction of the ovary to the ovulation stimulation, and as these women have no problem in ovarian response to ovulation stimulation, their mental health depends more on other factors . In addition, all subjects, who sustained the research were those who reached the stage of the harvesting process; the potential for ovum production was proven for these women . This would enhance the self - esteem of subjects and decrease their concern over ovarian nonresponsiveness . But direct correlation between the level of anxiety and depression in the stage after harvesting process with social impairment and hypochondriasis indicates that in this stage also the awareness of social dysfunction, independent from financial status, will have its own effect on mental health . Factors other than ovulation stimulation process which might influence the individuals levels of depression and anxiety have not been assessed in the present research . Depression and anxiety might result from a variety of factors, which require more extensive research . Another result of this research was that financial status, independent from the quantity of follicle obtained from ovulation stimulation, showed a reverse correlation with anxiety level after harvesting process . In ozken's research, also financial status was a factor determining the anxiety in infertile couples . Regarding financial pressures resulting from high costs of assisted reproduction treatments, which lead to the success of only 35% for each cycle, women's concerns over the success of results in fertilization as an outcome of infertility of the partner detected after harvesting process while insemination in laboratory are not far from expectation . Nevertheless, although women are healthy in terms of fertility in couples undergoing male factor infertility treatment, effort to have babies not by normal methods and facing difficult, costly treatment protocols might threaten their evaluation of their feminine role, thus reducing their self - esteem . Another finding of this research was the existence of correlation between depression level after treatment process and anxiety level at the first stage, while this correlation was observed between depression level after treatment process and anxiety level at the second stage . This finding indicates that women, who enter the process of treatment with a higher anxiety level are prone to a higher level of depression; therefore necessary measures should be taken to prevent it . One of this study's limitations was that mental health assessment was conducted after starting the primary stages of the treatment therefore, the stress of this stage could affect the mental health during the whole process . This study showed that fertile women who undergo assisted reproduction treatments do not start the treatment process in an appropriate mental health condition, and this condition would continue during the whole process until egg harvesting . Therefore, it is suggested that before starting the treatment the mental health of these women should be evaluated using screening tools and counseling sessions would be applied for vulnerable women.

Balance impairment is an important fall - risk factor, and increases in range of postural sway in the mediolateral direction in older adults are associated with increased fall - risk and rates . Postural sway has been shown in older adults to be strongly related to other measures of balance . Multivariate analysis reveals serum vitamin d levels as an independent variable associated with postural sway . In individuals with suboptimal levels of vitamin d, epidemiological studies have shown that vitamin d levels show seasonal variation [7, 8]. Lowest levels of serum vitamin d are recorded towards the end of winter, approximately four weeks after the shortest day of the year . Overall, vitamin d supplementation did not reduce rate of falls (rar 1.00, 95% ci 0.90 to 1.11; seven trials; 9324 participants) or risk of falling (rr 0.96, 95% ci 0.89 to 1.03; 13 trials; 26, 747 participants) but may do so in people with lower vitamin d levels before treatment . Older adults are at risk for lower levels of serum vitamin d because of age - related changes in uvb absorption and skin capacity to synthesize vitamin d, reduction in activation in the kidneys, and reduced expression of vitamin d receptors in tissues . There are many factors affecting fall - risk for older individuals, and although these may be different for inside and outside falls, strength and balance remain two important physical fall - risk factors . A recently published overview of the literature supports an assertion that age - related changes in postural reactions may be related to vitamin d status - mediated through either central nervous system integration or antigravity muscles as the effectors in postural responses . Despite changes in vitamin d across the seasons, muscle strength in the quadriceps muscles has been shown to remain stable . The relationship of postural sway to any potential increased winter fall rate and decreased levels of vitamin d has not been investigated . The winter season sees an increase in injuries from falls and in the number of accidental deaths from falls . Fracture rates from falls in older adults also increase at the end of the winter season, following two to eight weeks after the nadir in serum vitamin d levels . Some studies report an increased rate of falls, for both inside and outside falls [13, 14]; however, significant seasonal variation in fall rates was not found in a three - year study while, in a second study, seasonal variation in fall rates was reported in women but not in men . Coincident static balance changes with any potential increased fall rates in winter have not been previously reported . The data presented here forms part of a larger study, from which two other papers with the same clinical trial registration have been published [12, 16]. The aim of this study was to determine differences in static balance (postural sway), vitamin d, incidence of falls, and type of fall serially at the end of each season over a 12-month period, in older community - living adults . We hypothesised that postural sway, falls, and vitamin d would show a seasonal variation and that there would be an inverse relationship between vitamin d and the other variables . At the end of consecutive seasons, static balance, vitamin d status, and fall rate were measured within a longitudinal study design . No intervention was implemented by study researchers, so that the study could identify natural variations that occur over the seasons . Data was collected over a three - week period in each season from end of spring 2009 to the end of spring 2010, with collection of data timed to coincide with expected peaks and troughs in serum vitamin d levels in australia at latitude 41 degrees south (tasmania). After each assessment, participants were given an appointment for the next collection block in three months' time . Independently living community - dwelling adults aged between sixty and eighty - five years were recruited through local print media and community clubs . Daily intake of oral supplementation of vitamin d of greater than 800 international units was also an exclusion criterion . Participants were also excluded if they had a history of neurological disease and were withdrawn if they suffered a medical condition while participating in the study that would impact on their ability to perform the physical tests . Liver and kidney disease both impact vitamin d metabolism, and any potential participants with either of these conditions were excluded . A priori sample size calculation was based on a previous study reporting mediolateral sway range in a sample of community - dwelling older adults, and this indicated a minimum requirement of 81 completed participants (minimum effect size 2.5 mm sway; standard deviation (sd) 8 mm; power 0.8, alpha 0.05). Ninety - eight participants were recruited with the anticipation of a 15% drop - out rate . This repeated measures cohort study was designed to be able to detect differences in postural sway . This project received ethical approval from the human research ethics committee (tasmania) network (h0010561). Postural sway range in the medio - lateral sway direction was measured using a force platform (amti accugait pjb 101, massachusetts, usa) for thirty seconds under conditions of eyes open and closed, as well as with the additional challenge of using a 6.5 cm foam cushion (eyes open and eyes closed) (airex elite balance pad ag, switzerland). Participants were asked to remain stationary with their arms by their sides and look straight ahead while standing on the force platform . Foot position (bare feet) was standardised with heels 4 cm apart using marked placement for the feet to ensure repeatability between testing occasions . Venous blood samples were collected and clotted, then centrifuged (1610 relative centrifugal force) for 15 minutes . Serum concentration of 25-hydroxy vitamin d was measured by direct, competitive chemiluminescent immunoassay in a commercial accredited laboratory, using liaison method (diasorin inc ., participants received an individually coded calendar for 12 months of the study on which to report any falls and associated details by date, including information regarding the location and cause of the fall . Information about the type of fall, any injuries that resulted, and if medical attention was sought was recorded . This study utilised the who definition of a fall as an event which results in a person coming to rest inadvertently on the ground or other lower level . Annual cyclic trends were investigated by fitting a sine wave formula to data for postural sway and vitamin d, with the amplitude of the seasonal variation (in percentage change) and the annual mean values were estimated using repeated measures nonlinear regression, adjusted for age and gender . For secondary analysis the three monthly data were interpolated linearly to estimate intermediate values to correspond with the observed fall incident data . Mixed - methods poisson regression was used to determine associations between falls, fall injuries, and postural balance and vitamin d. the association between postural balance and vitamin d and season was estimated using mixed - methods linear regression, adjusted for age, gender, and strength . For comparison, seasonal data for falls was grouped into autumn and winter and compared to spring and summer . Physical activity using the champs questionnaire and muscle strength using the physiological profile assessment tools were recorded, and this information has been published elsewhere but forms an integral part of the meta - analysis of this paper . Data from eighty - eight participants (70% females) are included in the final analysis . Five people did not attend appointments, and five people could not complete testing because of medical events . The participants had a mean (sd) age of 69.2 (6.5) years and body mass index 27.4 (3.9) kg m. all participants were living in their own homes independently, with only 10% being sole occupants . Common chronic controlled health conditions included cardiovascular disease (39%) and arthritis (14%). Twenty - six percent of the participants reported the use of more than 4 medications . All four balance measures had the highest sway scores (poorest balance) at the first end of spring measurement . All other seasonal measures were significantly different from this first time point (all p <0.05), but no subsequent significant difference was seen after any other seasonal measures, indicating a lack of seasonal variation in this outcome (p> 0.05). No associations between postural sway and vitamin d were observed (all p> 0.05). Increased postural sway was associated with fall injuries (irr 1.59 (ci 1.14 to 2.24) (p = 0.007) but not fall rates (irr 1.36 (ci 0.95 to 1.97, p = 0.09). There was 15% variation in this variable over the year, with a peak at the end of summer and the lowest values at the end of winter . Seventy - five percent of fall diaries were posted on schedule while the remaining diaries were returned at the subsequent assessment appointment, resulting in a compliance of 100% . Thirty - three percent of the cohort (29 people) fell at least once, with 10% of the whole group falling multiple times (8 people). Over the duration of the study, 48 falls were recorded: 14 of these occurred inside the house, and 34 occurred outside . Six falls were due to fainting or dizziness and forty due to trip - related events, with one categorised as being pushed over (by a horse) and one not able to be categorised . Twenty - eight falls resulted in injury, with only four requiring medical treatment (including one fracture). Further details on season variation in location and type of fall are provided in table 2 . There were significantly fewer falls during spring than any other season (p = 0.01), with no other differences between the seasons recorded . When falls data were combined from autumn and winter seasons and compared to the combined spring and summer seasons, there were more falls reported in the combined autumn and winter seasons (30 compared to 18). Less injuries from falls were recorded in spring than any other season (p = 0.02), with no other seasonal differences recorded . This is the first cohort study to determine that no seasonal variation of postural sway occurs across 12 months in our population . Significant seasonal variation in serum vitamin d levels, with higher serum levels in summer, was recorded . There was a significant relationship between postural sway and the number of injurious falls observed, with lower values for sway range (i.e., better stability) associated with less fall injuries . A significant learning effect was seen with all measures of mediolateral postural sway with time point one having larger ranges than all of the other time points (all p> 0.05). Further to this, there was no seasonal variation in postural sway under any of the four static balance test conditions measured (eyes open and closed on a firm surface or foam surface). Postural sway range has been used to identify those people with balance impairment, and it may be useful in describing the fall - risk status of a particular individual . Our data indicates that postural sway does not appear to be subject to changes across the year within a participant . It has been suggested that this measure is important in describing sensorimotor deficits or disability rather than functional abilities and hence may not be subject to changes that may occur due to altered patterns of activity or sunlight exposure seen seasonally . The stability of this measure in a cohort across a year provides important information for researchers planning interventions designed to impact postural sway and clinicians who are measuring the effectiveness of their interventions . Mediolateral sway range has been shown to be an independent fall - risk factor for indoor falls . In our study, a lower proportion of falls occurred indoors (29%) compared to outdoors (71%), and although this is similar to other studies where more healthy samples have been reported as having a greater proportion of falls being outdoor falls (74%;), it may be a reason why no association between sway range and fall incidence was seen . A trend for this association was evident and a larger sample size may have found a significant relationship between these two variables, as this study was powered to determine mediolateral sway changes not fall rates . An association between increased sway range and rate of injurious falls was recorded in our study, reinforcing the importance of this measure for those most at risk of injury . Overall there was no significant relationship found between postural sway and vitamin d. as increased postural sway is linked to low levels of vitamin d, it may be that the levels of vitamin d were sufficient for this parameter even at their lowest levels in participants in this study and did not influence postural sway . If this threshold situation is true, it may be that seasonal variation in postural sway may be present in a population with much lower levels of vitamin d, but this is outside the scope of the current study . Annual rates of falling for adults over 65 have been reported up to 40%, and although our cohort includes some adults between the ages of sixty and sixty - five, with a mean age of 69 years and a fall rate of thirty - three percent, our population appears to be representative of older community - dwelling adults in terms of fall rate . Fall rates in older - old adults (over 75 years) have been shown to vary seasonally, but consistent data for the general population of healthy older community - dwelling adults has not been previously reported . One previous study grouped the peak seasons of winter and autumn together and found differences in fall rates . Manipulation of our data in a similar way reveals that there were more falls during the autumn / winter half year compared to spring / summer (table 2). These seasonal differences may be related to intrinsic factors that may be subject to seasonal variation (e.g., vitamin d, physical activity, and muscle strength) as well as seasonally related environmental factors (e.g., weather, temperature). Further research needs to investigate interventions to address potentially modifiable factors to reduce the increased falls risk in the autumn / winter period . Our data indicates a higher rate of falls in summer than has previously been reported, perhaps due to activity characteristics of our cohort . Summer and winter falls differ between the genders, with more men falling due to slips in winter and more women falling due to trips in summer . The high proportion of women in our study (79%) may be a factor in the high rate of summer falls observed . Another factor to consider is the relationship between fall status and vitamin d. although 60 nmol / l has been determined to be the cutoff for fall - risk function, 16/48 falls (33%) occurred in participants in this study whose vitamin d was above that cutoff level . This may be explained by the higher proportion of summer time falls observed in our study . During summer months, with longer hours of daylight at this latitude, a large proportion of the falls occurred in the outside (13 of the 14 falls 93%) (table 2). By contrast, in winter out - of - doors fall rate was reduced to 64%, indicating a higher winter time proportion of inside falls . For our generally healthy study population, it is likely that our participants were engaged in outdoor activities with higher associated risk of falls during the warmer weather; for example, several summer falls occurred while bushwalking . Fall injuries, especially fractures, have been found to increase in winter; this includes both inside (hip) and out - of - doors falls (wrist). Studies in the area of seasonal variation in fracture rates provide good evidence for increased fracture rates from falls in winter, but these appear in populations with older participants than ours (mean age over 75 years) [8, 13]. Our study recorded few injuries that required medical attention, hence making it difficult to compare serious fall injury data . Although this study aimed to recruit independently living community - dwelling older adults, bias in the sample may be present, as volunteers to this type of research project may be more robust than the community members at large . The lack of more frail subgroups, including those with cognitive impairment, depression, or using walking aids, limits the generalisability of this study . This study provides some evidence that measurements of postural sway may be affected by some effect of test - retest learning, and this needs to be considered in future research . This is the first study to investigate the effects of season and postural sway and found that postural sway remained stable over the 12 months . This study provides important evidence for clinicians and researchers that postural sway remains stable over an annual cycle but may be influenced by a learning effect.

Since the dawn of history, nature and natural sources, such as plants, animals, microbes, and minerals have remained a veritable source of bioactive compounds with medicinal values . Among these sources, plants have been the most explored and exploited for their bioactive medicinal components . Lead compounds or templates for the rational development of drugs are of more specific efficacies and fewer side effects (1). One of the botanicals of interest in which the abortifacient claims in the folk medicine have been substantiated by scientific evidence (2) but with no documentation in the open scientific literature on its bioactive abortifacient agent(s) is senna alata . Senna alata (linn .) Roxb (leguminosae), also known as craw - craw plant or ringworm plant (english), asunwon oyinbo (yoruba - western nigeria), nelkhi (igbo - eastern nigeria), filisko or hantsi (hausa - northern nigeria) is an erect tropical, annual herb which grows up to 0.15 m high . The fruit is a pod, while the seeds are small and square in shape . The plant has been claimed to be used in the management of several diseased conditions such as hepatitis, dermatitis, jaundice, gastroenteritis, eczema, constipation and diarrhoea (3, 4). Previous studies have shown that s. alata have antifungal, antibacterial and antioxidant activities (3, 58). Furthermore, yakubu et al . (2) reported that the aqueous leaf extract of the plant contained saponins (1.22%), flavonoids (1.06%), cardiac glycosides (0.20%), phenolics (0.44%), alkaloids (0.52%), cardenolides and dienolides (0.18%). Aqueous leaf extract of s. alata has also been scientifically validated for its acclaimed use as an abortifacient (2). However, there is no study in the open scientific literature that has reported on the exact bioactive abortifacient agent(s) in s. alata leaves . Therefore, the present study aimed to validate the speculation that the alkaloids in aqueous extract of s. alata leaves are responsible for its abortifacient activity . The focus on alkaloid was a follow - up from our previous study that speculated on alkaloids to be responsible for the abortifacient activity of the crude extract of s. alata leaves and from several studies that implicated the phytochemical property of colchicine, quinazoline alkaloids, e.g. Vasicine and vasicinone in several botanicals such as xylopia aethiopica, peganum harmala epigeal parts, areca catchu nuts and gloriosa superba roots as abortifacient bioactive agents and/or their role in the contraction and relaxation of uterine muscles (912). The plant leaves, obtained from herb sellers at oja tuntun, (new market) in ilorin, nigeria, was authenticated at the herbarium unit of the forestry research institute of nigeria (frin) in ibadan, nigeria . A voucher specimen (fhi 10845) was deposited at the herbarium of the institute . Assay kits for both glucose and cholesterol were products of randox laboratories, ltd, united kingdom, while those of progesterone, follicle stimulating and luteinizing hormones were products of inteco uk ltd, united kingdom . Thin layer chromatographic (t lc) plates and silica gel were products of merck (darmstadt, germany). Para - nitrophenyl phosphate and other reagents were products of sigma- aldrich inc ., st . Male and female wistar rats (rattus norvegicus) weighing 178.913.07 and 143.99 1.21 g, respectively, were obtained from the animal holding unit of the department of biochemistry, university of ilorin, ilorin, nigeria . The animals which were housed individually in plastic cages and placed in a well - ventilated room (temperature: 28 - 31c; photoperiod: 12 hr natural light and 12 hr darkness; humidity: 50 - 55%) were provided with unrestricted access to rat pellets (bendel feeds and flour mills, ewu, nigeria) and water . The animals were also handled according to the guidelines of the european convention for the protection of vertebrate animals used for experimental and other scientific purposes ets 123 (13). The leaves of senna alata which were oven - dried at 40c for 48 hr were pulverised using a mikachi blender (mk-1830, china). Alkaloids were extracted from the powder according to the procedure described by manske (14). A known amount (500 g) of the powder was extracted in 1.2 l of hexane for 72 hr and filtered with whatman no the hexane extract (containing fats, oils, terpenes, waxes) were discarded and the resulting residue extracted again in 1.2 l of methanol for a week and subsequently filtered . The filtrate was evaporated using a rotavapor (r110, gallenkamp, england, uk) and the process was repeated two more times . The three filtrates were combined, concentrated to give a methanolic, green slime (90 g) which was treated with 1 m hcl and then basified by adding 5 m naoh with continuous stirring until a cloudy precipitate appeared . A known volume (500 ml) of chloroform and 200 ml of 1 m nacl were added and the process was repeated three more times . Equal volumes (150 ml) of 1 m nacl and 5 m naoh were added to the organic layer in a separating funnel after which the mixture was evaporated to yield brownish - black slurry (18 g) of alkaloids that corresponded to a yield of 3.60% that was used for subsequent experiments . The procedure described by singh and sahu (15) was adopted for the preparation of thin layer chromatography (tlc) plates . Furthermore, about 10 l of the test solution (extract) was spotted onto the thin layer plate using a micropipette after which the plates were developed in chloroform: methanol (10:2) in which 0.01 g / ml of butylated hydroxyl toluene and butylated hydroxyl anisole were added to prevent oxidation (which may lead to increase in the number of bands with time) (16). The relative values related to the solvent front (rf) of the spots were also computed . Twenty - four pregnant rats were allocated in a complete randomize design to four groups (a, b, c and d) consisting of six animals each . Animals in group a (controls), orally received 0.5 ml of distilled water with the aid of an oropharyngeal cannula while those in groups b, c and d orally received 0.5 ml of the alkaloids corresponding to 250, 500 and 1000 mg / kg body weight, respectively . The administration was done once daily from day 10 until day 18 of pregnancy (period of organogenesis in wistar rats) (18). By the 19th day (24 hr after their last dose) the following parameters were recorded / computed: number of live fetuses; number of dead fetuses; average weight of live foetuses; survival ratio (%) = (number of live fetuses/ number of live + dead fetuses) 100; number of rats that aborted; percentage of rats that aborted= (number of rats that aborted / number of rats assessed) 100; number of rats with vaginal bleeding; number of implantation sites; number of corpora lutea; implantation index=(total number of implantation sites / number of corpora lutea) 100; pre - implantation loss=(number of corpora lutea number of implantation sites / number of corpora lutea) 100; post - implantation loss= (number of implantation sites number of live fetuses / number of implantation sites) 100; number of resorption sites = number of implantation sites in the control animals number of implantations in the test animals; resorption index=(total number of resorption sites / total number of implantation sites) 100 . The weights of the animals both before pairing and prior to sacrifice, as well as feed and water intake were also recorded . The in vivo estrogenic / anti - estrogenic response of the rats to the alkaloids was evaluated by adopting the procedure described by kanno et al . Twenty - four ovariectomized, female rats (159.677.12 g) were allocated to four groups (a, b, c and d) of six animals per group . Group a (controls), orally received 0.5 ml of distilled water while animals in groups b, c and d orally received 0.5 ml of the alkaloids corresponding to 250, 500 and 1000 mg / kg body weight, respectively . The administration which commenced from the eighth day of ovariectomy, lasted for another seven days . On day 16, the weights of the animals were determined prior to sacrifice and uterine to body weight ratio was computed . The uterine protein, glucose, cholesterol, and alkaline phosphatase activity were determined using standard procedures (2023). The serum and uterine homogenates were prepared according to the procedures described by yakubu and bukoye (24). The procedures outlined in the manufacturer's protocol were adopted for the quantitative determination of progesterone, follicle stimulating and luteinizing hormones in the serum of the animals . Data which were expressed as the meansd of six independent replicates were statistically analyzed using one - way analysis of variance and duncan multiple range test . The plant leaves, obtained from herb sellers at oja tuntun, (new market) in ilorin, nigeria, was authenticated at the herbarium unit of the forestry research institute of nigeria (frin) in ibadan, nigeria . A voucher specimen (fhi 10845) was deposited at the herbarium of the institute . Assay kits for both glucose and cholesterol were products of randox laboratories, ltd, united kingdom, while those of progesterone, follicle stimulating and luteinizing hormones were products of inteco uk ltd, united kingdom . Thin layer chromatographic (t lc) plates and silica gel were products of merck (darmstadt, germany). Para - nitrophenyl phosphate and other reagents were products of sigma- aldrich inc ., st . Male and female wistar rats (rattus norvegicus) weighing 178.913.07 and 143.99 1.21 g, respectively, were obtained from the animal holding unit of the department of biochemistry, university of ilorin, ilorin, nigeria . The animals which were housed individually in plastic cages and placed in a well - ventilated room (temperature: 28 - 31c; photoperiod: 12 hr natural light and 12 hr darkness; humidity: 50 - 55%) were provided with unrestricted access to rat pellets (bendel feeds and flour mills, ewu, nigeria) and water . The animals were also handled according to the guidelines of the european convention for the protection of vertebrate animals used for experimental and other scientific purposes ets 123 (13). The leaves of senna alata which were oven - dried at 40c for 48 hr were pulverised using a mikachi blender (mk-1830, china). Alkaloids were extracted from the powder according to the procedure described by manske (14). A known amount (500 g) of the powder was extracted in 1.2 l of hexane for 72 hr and filtered with whatman no . 1 filter paper . The hexane extract (containing fats, oils, terpenes, waxes) were discarded and the resulting residue extracted again in 1.2 l of methanol for a week and subsequently filtered . The filtrate was evaporated using a rotavapor (r110, gallenkamp, england, uk) and the process was repeated two more times . The three filtrates were combined, concentrated to give a methanolic, green slime (90 g) which was treated with 1 m hcl and then basified by adding 5 m naoh with continuous stirring until a cloudy precipitate appeared . A known volume (500 ml) of chloroform and 200 ml of 1 m nacl were added and the process was repeated three more times . Equal volumes (150 ml) of 1 m nacl and 5 m naoh were added to the organic layer in a separating funnel after which the mixture was evaporated to yield brownish - black slurry (18 g) of alkaloids that corresponded to a yield of 3.60% that was used for subsequent experiments . The procedure described by singh and sahu (15) was adopted for the preparation of thin layer chromatography (tlc) plates . Furthermore, about 10 l of the test solution (extract) was spotted onto the thin layer plate using a micropipette after which the plates were developed in chloroform: methanol (10:2) in which 0.01 g / ml of butylated hydroxyl toluene and butylated hydroxyl anisole were added to prevent oxidation (which may lead to increase in the number of bands with time) (16). The relative values related to the solvent front (rf) of the spots were also computed . Twenty - four pregnant rats were allocated in a complete randomize design to four groups (a, b, c and d) consisting of six animals each . Animals in group a (controls), orally received 0.5 ml of distilled water with the aid of an oropharyngeal cannula while those in groups b, c and d orally received 0.5 ml of the alkaloids corresponding to 250, 500 and 1000 mg / kg body weight, respectively . The administration was done once daily from day 10 until day 18 of pregnancy (period of organogenesis in wistar rats) (18). By the 19th day (24 hr after their last dose) the following parameters were recorded / computed: number of live fetuses; number of dead fetuses; average weight of live foetuses; survival ratio (%) = (number of live fetuses/ number of live + dead fetuses) 100; number of rats that aborted; percentage of rats that aborted= (number of rats that aborted / number of rats assessed) 100; number of rats with vaginal bleeding; number of implantation sites; number of corpora lutea; implantation index=(total number of implantation sites / number of corpora lutea) 100; pre - implantation loss=(number of corpora lutea number of implantation sites / number of corpora lutea) 100; post - implantation loss= (number of implantation sites number of live fetuses / number of implantation sites) 100; number of resorption sites = number of implantation sites in the control animals number of implantations in the test animals; resorption index=(total number of resorption sites / total number of implantation sites) 100 . The weights of the animals both before pairing and prior to sacrifice, as well as feed and water intake were also recorded . The in vivo estrogenic / anti - estrogenic response of the rats to the alkaloids was evaluated by adopting the procedure described by kanno et al . Twenty - four ovariectomized, female rats (159.677.12 g) were allocated to four groups (a, b, c and d) of six animals per group . Group a (controls), orally received 0.5 ml of distilled water while animals in groups b, c and d orally received 0.5 ml of the alkaloids corresponding to 250, 500 and 1000 mg / kg body weight, respectively . The administration which commenced from the eighth day of ovariectomy, lasted for another seven days . On day 16, the weights of the animals were determined prior to sacrifice and uterine to body weight ratio was computed . The uterine protein, glucose, cholesterol, and alkaline phosphatase activity were determined using standard procedures (2023). The serum and uterine homogenates were prepared according to the procedures described by yakubu and bukoye (24). The procedures outlined in the manufacturer's protocol were adopted for the quantitative determination of progesterone, follicle stimulating and luteinizing hormones in the serum of the animals . Data which were expressed as the meansd of six independent replicates were statistically analyzed using one - way analysis of variance and duncan multiple range test . The alkaloids yielded 0.30 g which corresponds to 1.50% of the starting material of 500 g. the five different spots gave rf values of 0.28, 0.33, 0.39, 0.47 and 0.55 . The spots gave positive reaction with both meyer's and wagner's reagents producing a creamy precipitate and reddish - brown spots on a grey background (tlc). The alkaloid truncated the development of the fetuses as none of them survived in the experiment groups as against the average number of live fetuses of 10.26 in the distilled water - treated control animals (table 1). The average weight of live fetuses in the controls was 4.92 g against none in all the extract - treated animals . The percentage of fetal death in the 250, 500 and 1000 mg / kg body weight of the alkaloid - treated animals was 6.03, 6.00 and 6.50, respectively . There was neither episode of abortion nor vaginal bleeding in all the alkaloid - treated animals . Both the number of implantation sites and corpora lutea decreased significantly (p <0.05). While the implantation index was similarly high in all the alkaloid - treated animals, the resorption index, as well as pre- and post- implantation losses were many fold higher than the controls (table 1). Although, all the pregnant rats gained weight at the end of the experimental period (final maternal weight) compared with their weight prior to pairing with the males (initial maternal weight) (table 1), the weight gained by the alkaloid - treated animals was about 50% less than the control animals . Furthermore, the feed and water intake by the animals treated by different doses of the alkaloid decreased significantly (p <0.05). Effect of the alkaloids from senna alata leaves on some abortifacient parameters of pregnant rats values are expressed as meansd of six independent determinations; test values carrying superscripts different from the control for each parameter in the same row are significantly different (p <0.05). Maternal weights of the animals before pregnancy were compared with their corresponding weights after pregnancy for each treatment group (p <0.05) all different doses of the alkaloids significantly (p <0.05) decreased the serum concentrations of the follicle stimulating hormone, luteinizing hormone and progesterone in the pregnant animals (table 2). Effect of alkaloids from senna alata leaves on some female reproductive hormones of pregnant wistar rats values are expressed as meansd of six independent determinations; test values carrying superscripts different from the control for each parameter in the same row are significantly different (p <0.05) the alkaloids also decreased (p <0.05) the absolute weight of the uterus, computed uterine to body weight ratio, and concentrations of uterine glucose and cholesterol (table 3). In contrast, the concentration of uterine protein and the activity of alkaline phosphatase increased significantly (p <0.05). Furthermore, the alkaloids did not provoke vaginal opening or cornification in any of the animals (table 3). Effect of alkaloids from senna alata leaves on some indices of oestrogenicity in pregnant rats values are expressed as meansd of six independent determinations; test values carrying superscripts different from the control for each parameter in the same row are significantly different (p <0.05) analysis of the tl chromatogram indicated that the mixture consisted of five alkaloids as evidenced by the creamy precipitates and reddish- brown spots produced with meyer's and wagner's reagents, respectively . In the present study, alkaloids from s. alata leaves significantly affected the fetal and maternal parameters of the animals . For instance, the death of the fetuses in the alkaloid - treated pregnant rats may not only suggest inhibition of mitotic division of the fetuses (25), since the animals were exposed during the period of organogenesis, but may also show its relevance to the reduction in the concentrations of gonadotropins and progesterone in the present study . (2) on the aqueous leaf extract of the plant where only the 500 and 1000 mg / kg body weight of the extract produced fetal death . Furthermore, the absence of abortion and vaginal bleeding in the alkaloid - treated animals which were hitherto observed with the crude extract in our previous study suggest zero abortifacient activity for the alkaloid . Therefore, the abortifacient activity of the aqueous extract of the plant leaf reported earlier (2) is not due to the alkaloidal content alone, but to some other phytochemicals such as saponins and flavonoids which may act synergistically or additively to produce the desired result . It is interesting to note that both the number of implantation sites and corpora lutea decreased in the alkaloid - treated animals when the process of implantation ought to have been completed (implantation takes place normally within 5 - 6 days post - coitus in rats) before the exposure of the animals to the alkaloid mixture (treatment commenced from day 10 of pregnancy). The reason for this decrease is not immediately known but may not be unconnected with the consequence of some general hormonal effect (reduced progesterone) and/or absence of conceptuses growth (26). It is also possible that blastocytes were not activated and well - positioned before implantation, probably due to impaired muscular activity of the uterus (27). The implantation index and pre - implantation loss evaluates the number of blastocysts implanted in the uterus while the resorption index and post - implantation loss relate to the number of implanted blastocysts and those that have not developed (18, 28). Therefore, the high implantation index, and pre- and post- implantation losses suggest that pregnancy was interrupted by the alkaloid, probably creating an environment that was not conducive for the fertilized eggs . Normally, an abortion in most cases is accompanied by vaginal bleeding and when this is absent, the resorption will increase . Therefore, the increase in the resorption index in the alkaloid- treated animals confers antifertility effects (such as anti - implantation, anti - blastocystic and antizygotic) of the alkaloid . The alkaloid did not exhibit complete abortifacient effect since the pregnant animals exposed to the alkaloid presented with closed vagina just like the controls . The findings in the present study is similar to the report by elbetieha et al . (29) where the administration of 200, 400 and 800 mg / kg body weight of ethanolic extract of salvia fruticosa did not cause pregnancy failure but increased the number of resorption in the pregnant rats . The alkaloids reduced the sense of taste and appetite of the animals as evidenced by the decrease in feed and water intake . Such reduction may account for the decrease in the computed percentage gain of maternal weight and may also be a consequence of impaired growth and development of the uterine contents (30). All these findings except those of feed and water intake, as well as maternal weight gain, contrast the previous report by yakubu et al . It is well - known that for the implantation of the fetus and sustenance of pregnancy, an exact equilibrium must exist between the secretion of estrogen and progesterone; this regulation is controlled by luteinizing and follicle stimulating hormones (31). Thus, the reduction in the gonadotropins may equally be responsible for the reduced concentration of progesterone in the present study and this may not only account for the death of the fetuses, but also for the increase in the resorption sites . The reduction in progesterone may suggest impaired endometrium function, which will adversely affect the normal secretion of special proteins required to nourish an implanted fertilized egg, and consequently, pregnancy failure . Thus, it is possible that the alkaloids posses anti - gonadotropic and anti - progestogenic activities which are inimical to the continued development of the fetuses . Many plant extracts with anti - fertility properties are known to exhibit estrogenic activity by increasing protein synthesis, uterine weight, water uptake and retention of fluid leading to ballooning of the uterus, uterine content of glucose, cholesterol, glycogen and alkaline phosphatase activity, thereby changing the uterine milieu and creating non - receptive conditions in the uterus (32). Therefore, the contrasting effects the alkaloids on some of these parameters in the present study suggest that estrogenic activity was not total but selective . The alkaloids exhibited more anti - oestrogenic activity (71.43%) than oestrogenic (28.57%). Overall, the alkaloid from s. alata leaves at the oral doses of 250, 500 and 1000 mg / kg body weight on daily basis from days 10 until day 18 post - coitum exhibited several potential effects on the maternal and fetal outcomes of pregnant rats (anti - implantation, anti - gonadotropic, anti - progesteronic, selective estrogenic, embryonic resorption and fetotoxic activities), but it could not induce abortion in the animals . Therefore, the alkaloids may not alone be responsible for the abortifacient effects of the crude extract of s. alata which had been reported earlier . Finally, work is in progress on isolating the other phytochemicals (saponins and flavonoids) and evaluating their synergistic effects in pregnant animals.

Erg protein expression has been recently suggested to be reflective of erg gene rearrangements in prostate cancer (pca) documenting remarkable concordance between the two [16]. The rearrangements between the androgen receptor - regulated gene tmprss2 (21q22.3) and members of the ets family member of transcription factor gene, most commonly erg (21q22.2), are among the most common genetic alterations detected in prostate cancer [711]. Erg gene rearrangements have been detected in roughly half (4060%) of pca of surgical cohorts compared to a rate of 12%15% in incidental or watchful waiting cohorts [7, 1218]. Previous studies investigating the prognostic significance of erg gene rearrangements have revealed mixed results [1922]. However, it is becoming more evident that erg gene rearrangements signify a molecular subtype of pca . Some studies investigating the significance of erg protein expression in localized pca failed to show association with adverse clinical outcome [23, 24]. However, a recent report by our group demonstrated an association of erg expression with lethal disease in patients with unsuspected and advanced / castrate resistant disease who were treated by transurethral resection of prostate . Moreover, we documented significant association between erg expression and both gleason score and tumor volume . Studies from our group and others have also linked erg status to responsiveness to hormonal therapy, and longer progression time to castration resistant disease, compared to men with no erg expression [24, 25]. In the current study, we investigated the association of erg protein expression to clinical - pathological parameters in a cohort of men with localized prostate cancer . The study cohort consisted of 198 patients who were treated by retropubic radical prostatectomy for localized prostate cancer with a mean followup of 4.8 years (range 015.8). Clinical progression was defined as a postoperative serum psa elevation of> 0.2 ng / ml . Prostate samples were embedded onto three tissue microarray (tma) blocks using a manual tissue arrayer (beecher instruments, silver spring, md, usa). One to nine cores (average 3.3), 0.6 mm in diameter, were sampled including benign, high grade intraepithelial neoplasia (hgpin), and prostate cancer (pca). After construction, 4 m sections were cut and stained with haematoxylin and eosin on the initial slides to verify the histological diagnosis . Briefly, 4 m thick sections from formalin - fixed paraffin - embedded tissue blocks were stained with ventana autostainer . Prior to the staining, heat - induced antigen retrieval was carried out by vegetable steamer in sodium citrate antigen retrieval buffer (10 mm ph 6.0) for 40 minutes and then cooled down to room temperature for about 20 minutes . The slides were incubated for 60 minutes at 37c with erg rabbit monoclonal antibody (epitomics, clone epr 3864) at 1: 50 dilution . A ventana iview dab detection kit (ventana tucson, az, usa) the diagnoses of all tma cores were confirmed by the three study pathologists (lht, cw, and tab). For each patient, the two predominant patterns were sampled and included on the tmas for analysis . Erg protein expression was assessed semiquantitatively using 3-tiered system (0, negative; 1, low; 2, high). Cases with either 1 or 2 intensity were considered positive based on previous correlation with erg gene rearrangement as detected by fluorescent in situ hybridization (data not shown). The erg antibody was consistently strongly expressed in endothelial cells, which acted as internal control for expression and intensity level . Patient characteristics were presented as frequencies and percentages for categorical variables and as means and ranges for continuous variables . Chi - square tests were used to test for associations between erg protein expression and gleason score, surgical margin, and pathological stage . The kaplan - meier approach along with the log - rank test was used for the survival analyses to test the association between erg expression and serum psa relapse . In all statistical tests, mean patients' age of this cohort was 64 years (range 42.780.5 years) with average follow - up time of 4.8 years (range 0.015.8 months). Table 1 demonstrates patients' demographics of the study cohort with respect to erg expression . Overall, there were no significant differences between the two subgroups (erg pos & erg neg) of patients except for pathological stage, with 37% of erg positive tumors detected in pt3 versus 24% in pt2 . To investigate erg expression in different diagnostic categories, we characterized erg expression based on individual cores sampled . When we accounted for foamy type pca morphology, the rate of erg expression was 15/84 (17.9%), compared to 302/704 (42.9%) in acinar pca (cases with no foamy type morphology) (p <0.001). There was no difference in high erg intensity between foamy type and acinar pca (data not shown). However, mean intensity level of erg in acinar pca was significantly higher than foamy type pca, 1.01 1.27 versus 0.37 0.83 (p <0.001). Erg intensity levels in hgpin were comparable to those in foamy type pca but slightly lower (0.13 0.56) but significantly lower than those in acinar pca (p <0.001) (figure 1). Erg expression was noted in 106/280 (37.8%), 175/463 (37.8%), and 37/108 (34.2%) of gleason scores 6, 7, and 810, respectively . Figure 2 demonstrates examples of erg expression in tissue samples and the distribution of erg in relation to gleason score . When investigating relations between erg expression and pathological parameters, there was significant association between erg expression and higher disease stage . In this cohort, erg expression was present in 50/131 (38.1%) patients with pt2 versus 30/55 (54.5%) patients with pt3 (p = 0.04). A similar association was also noted between erg expression and extra - capsular extension . In this cohort, 52/134 (38.8%) erg positive patients demonstrated organ confined disease versus 29/53 (54.7%) erg positive patients showing extra - capsular extension (p = 0.04). Similar trends were noted between erg expression and seminal vesicle invasion, but this was not statistically significant (p = 0.10) (data not shown). No significant association was noted between overall erg positivity and positive surgical margins (table 1). Similarly, no association was observed with postsurgical psa levels when assessed by univariate or multivariate analysis (figure 3). Although not informative due to limited patients' numbers, the association between erg expression and higher stage disease was more pronounced in patients with higher gs . In this cohort, none of the patients with gs> 7 and erg positive (0/7) were of stage pt2 compared to 47% (7/15) of erg negative patients who were of pt2 stage (p = 0.02) (table 2). This study reports on the potential significance of erg protein expression in localized prostate cancer . Erg gene rearrangements and erg expression have been documented in roughly 50% of localized prostate and locally advanced castrate resistant prostate cancer compared to 12%15% in watchful waiting or incidental cohorts [16, 17, 22, 25, 29]. Published reports on the significance of erg expression to patients' outcome are conflicting with some showing association with adverse outcome, while others document no association . Some suggest that it indicates a better prognosis [16, 22, 25, 2933]. However, it is proposed that erg signifies a molecular class of prostate cancer and may play a role in disease progression within those tumors . This pathway is closely linked to increased rate of pten genomic deletions as well as increased erg expression or erg gene rearrangements [34, 35]. Erg gene rearrangements and erg expression are associated with adverse outcome and lethal disease in watchful waiting or expectant cohorts [16, 25]. Moreover, patients with erg overexpression demonstrated shorter progression times to castrate resistance, needing surgical intervention (channel turp). In localized prostate cancer, the majority of the reported data suggest no prognostic implication for erg rearrangements or erg overexpression in relation to psa relapse or the recurrence of local disease [22, 36, 37]. However, two earlier reports suggested adverse association of erg rearrangements with psa relapse after radical prostatectomy [20, 31]. In nam's study, yashimoto's group noted that the adverse prognostic association was linked to pten genomic deletions . Of note, a study by reid et al . Documented that erg gene rearrangements in addition to pten genomic deletions had actually favorable outcomes compared to pten deletion alone . These observations suggest that the method utilized in determining erg status, the inclusion or exclusion of other genomic aberration, and the cohort chosen may be the reason for the different results obtained in various studies investigating the role of erg in prostate cancer . Our results support previous observations showing no prognostic relationship between erg overexpression and clinical outcome in localized prostate cancer . However, in contrast to earlier studies, we document significant association between erg expression and some pathological parameters . In this cohort, patients whose tumors were erg positive were at higher risk of exhibiting extra - prostatic extension and increased disease stage compared to patients whose tumors did not express erg . Specifically, all patients with gs> 7 and erg expression showed pt3 stage disease compared to none of pt2 . However, this data is limited by the number of patients within each gs subgroup and need further confirmation . More importantly, in addition to not documenting any clinical prognostic significance for erg expression, we did not observe any association between erg expression and other pathological parameters such as gleason score or surgical margins, which further diminishes any potential prognostic significance for erg expression, at least in localized pca . Another issue worth mentioning regarding erg is that, erg gene rearrangements have been previously associated with specific histopathological features and detected more frequently in some morphologic variants of prostate cancer than others . In this study, we confirm lower rates of erg expression in tumors with foamy / xanthomatous morphology which demonstrated lower mean expression intensities compared to acinar pca . The significance of this is not yet known, but suggests that those two types of tumors may be different at the molecular level . In conclusion, this study demonstrates significant association between erg expression and extra - prostatic extension and higher pathological stage in localized prostate cancer . Moreover, the lack of association with any other pathological parameters also significantly diminishes any potential clinical application of erg expression, at least in men with localized prostate cancer.

The three main arteries of the leg, the anterior tibial artery (ata), posterior tibial artery and peroneal artery, form a dense vascular network around the distal leg, ankle, and foot, which ramifies perforators as the basis of all kinds of pedicle flaps such as lateral or medial malleolar perforator flaps . There is plenty of literature describing the anatomy and clinical application of perforators of the peroneal artery and posterior tibial artery around the distal lower leg . However, literature regarding perforator flaps of ata or anterior supramalleolar artery (asma), one of the major branches of distal ata, are scant . These papers either mention the existence of the perforators from ata or asma, or describe their communication with the medial and lateral supramalleolar arteries . None of them have a detailed anatomical study on asma . The objective of this study was to identify the anatomic parameters of the asma and its perforators that would enable microsurgeons to harvest potentially multi - paddle flaps or composite flaps from the foot and ankle region . We try to preserve the ata intact after we harvest the composite flaps around the ankle using asma . We also want to use the ata as the only one pedicle and harvest multi - paddle flaps, decreasing the microsurgical risk greatly and minimizing the sacrifice of the blood supply to the ankle and foot . After the study was approved by the university of louisville irb; 24 lower extremities of fresh human cadavers were studied . The ata was cannulated and injected with 20 ml of red latex (microfilm, flow tech, inc .,) at the mid - point of lower leg where ata can be identified between the extensor digitorum longus and tibialis anterior muscles . Then, careful dissection along the distal 1/3 of the lower leg, the ankle, and the proximal foot (8 - 10 cm above the lateral malleolus and 1 - 2 cm anterior to the edge of the fibula) was performed to observe the origin and the course of the asma . The distance between the origin of asma and the extensor retinaculum (o - r) was recorded . The diameters of the asma proper as well as its branches (a set of fixed - width hooks was used for measurement), and the length of asma were measured . The collaterals between asma and the medial and lateral supramalleolar arteries and the number of perforators were observed . Chi - square test was used for this study to analyse the variation of the o - r . Fasciocutaneous flaps with multiple skin paddles were elevated based on the perforators to demonstrate its potential application . We classified the asma into four types according to the pattern of origin from the ata [figures 1 - 4]. Type a (n = 10) asma originates from the ata with a 1 - 1.5 cm long main trunk and then gives off its lateral and medial branches [figure 1a and b]; each consists of 2 - 3 perforators . In type b (n = 7), the asma does not have a main trunk, and its lateral and medial branches stem directly from ata with the sites of origin at the same level [figure 2a and b]. Type c (n = 6) is similar to type b, with the lateral and medial branches arising from the ata but at different origins [figure 3a and b]. Type d (n = 1) has 2 medial, and 1 lateral branch arising from the ata [figure 4a and b]. (a) type a the anterior supramalleolar artery originates as an artery stem . (1) short artery stem; (2) lateral branches; (3) medial branches; (4) descendent perforators; (5) ascendant perforators . (6) proximal end of anterior tibial artery (ata); (7) distal end of ata; (8) blood supply to tibia; (9) extensor retinaculum (b) the anterior supramalleolar artery originates as a 1 - 1.5 cm short artery stem and then ramifies lateral and medial branches . The medial branch gives off ascendant perforators and descendent perforators (a) type b the anterior supramalleolar artery originates as lateral and medial artery branches at the same point . (1) medial branches; (2) ascendant perforators . (3) descendent perforators; (4) lateral branches; (5) proximal end of anterior tibial artery (ata); (6) distal end of ata; (7) extensor retinaculum; (8) blood supply to tibia (b) the anterior supramalleolar artery orients as lateral and medial artery branches at the same point . The medial branch gives off ascendant perforators and descendent perforators (a) type c the anterior supramalleolar artery originates as lateral and medial artery branches at the different point . (1) medial branches; (2) ascendant perforators . (3) descendent perforators; (4) lateral branches; (5) proximal end of anterior tibial artery (ata); (6) distal end of ata; (7) extensor retinaculum (b) the anterior supramalleolar artery orients as lateral and medial artery branches at different point . The medial branch gives off ascendant perforators and descendent perforators (a) type d - the anterior supramalleolar artery originates as lateral and two medial artery branches at the different point . (1) medial branches; (2) lateral branches; (3) proximal end of anterior tibial artery (ata); (4) distal end of ata; (5) extensor retinaculum (b) the anterior supramalleolar artery orients as one lateral and two medial arteries branches at different point the mean distance between the origin of the proximal branch of asma and the superior margin of the extensor retinaculum (o - r) was 2.0 0.8 cm . The mean diameters of the medial branch (d1), lateral branch (d2) and the main trunk of the asma (d3) (only in type a) were 1.0 0.2 mm, 0.8 0.3 mm and 1.1 0.2 mm, respectively . The mean feasible length of the pedicles of the lateral flap (l1) and medial flap (l2) were 5.1 1.0 cm and 3.7 0.6 cm, respectively . Bi - foliate fasciocutaneous flaps can be harvested based on the medial and lateral branches of the type a asma with preservation of the ata [figure 5]. Data of 24 specimens of the anterior supramalleolar artery the biofoliate flap using type a anterior supramalleolar artery huber dissected 200 feet and was the first to describe the asma as a vessel arising about 5 cm above the ankle joint . Wee described that this septocutaneous branch of ata runs through the anterior crural septum between the tendons of tibialis anterior and extensor hallucis longus and gives out 3 perforators . Further named the reverse - flow flap based on the perforators of this artery as anterior tibial type iii . Later research showed that asma is always found in the distal 1/3 of the lower leg and appears 8 - 10 cm above the lateral malleolus, 1 - 2 cm anterior to the edge of the fibula . In 2003, koshima dissected 4 ankles and described that the asma has 1 - 2 perforators just above the extensor retinaculum and gives off the anterolateral and anteromedial branches . However, we still lack detailed anatomical knowledge of its origin, calibre and so on . The diameters of the lateral and medial branches of asma are 0.8 0.3 mm and 1.0 0.2 mm, respectively . The feasible pedicle lengths of these two branches are 5.1 1.0 cm and 3.7 0.6 cm, respectively . Thus, an island flap can be harvested based on one or both of the branches for defect coverage around the ankle . In addition, due to its collateral connection with the posterior tibial artery and peroneal artery, a reverse - flow anterolateral or anteromedial flap can also be designed as described in literature . The flaps with asma are very useful especially for patients who suffer from refractory malleolar ulcers due to paralysis, diabetes mellitus, venous stasis, or vasculitis . For patients who have cosmetic consideration, perforator - based adipofascial flap is also a good option with the use of asma . The perforators of the asma are suitable for a bi - foliate design in situations such as simultaneous defects of the volar and dorsal aspects of the hand . The flap based on asma is pliable and thin, which makes it an excellent option for hand and wrist coverage . If there is a simultaneous metacarpal deficiency at the same time, which can present especially in high - energy injuries, vascularised bone graft can also be harvested easily supplied by 1 - 2 perforators according to research by gilbert et al . An additional advantage of this perforator flap is that the ata can be preserved in type a asma, and morbidity of the donor site can be minimised . Based on different types of asma, its lumen size can vary from 0.8 0.3 to 1.1 0.2 mm, which is still suitable for microvascular anastomosis . A difficult problem that a hand surgeon encounters conventional methods include inserting the hand into an abdominal pocket for several weeks followed by skin graft, using a large flap to cover all the defects together, or covering the wounds with several free flaps at the same time . Often, these patients will still exhibit stiff digits and a bulky hand . And combined multiple single flaps would increase the surgical risk exponentially . In this instance, we propose a novel idea of using a multiple - foliate flap based on different perforators of the lateral and medial branches of the asma to cover multiple digits at the same time . These branches all derive from the same trunk - the ata and its terminal branch . This design provides the capability of harvesting up to 2 - 3 skin paddles based on one sizable pedicle, the ata . The terminal branches of the peroneal artery to ankle and foot, as well as the branches of posterior tibial artery to ankle and foot are also preserved . In this way, the vascular network of the ankle and foot is still preserved, and donor site morbidity is significantly decreased . There are several factors which should be kept in mind during the clinical application of asma based flaps . When the skin is opened, the lateral and medial branches of the asma are generally visible . If we want only the stem artery and their branches, we can just follow the course of these branches and easily find the origin of these branches . . However, in any situation it would be more effective to repair the extensor retinaculum carefully and immobilize the foot for 3 - 4 weeks . Further study and long - term follow - up are needed to understand the blood supply of the ankle and foot after harvesting the asma - based flap . Its sizable diameter and lengthy pedicle make it suitable as bi - foliate fasciocutaneous flaps or composite flaps.

Ankle dorsiflexion passive range of motion (df prom) measurements are performed in the field of physical therapy to estimate ankle motion during functional activities1 and to prevent lower extremity injuries2 . Although in the clinical setting, ankle df prom is frequently measured under non - weight - bearing (non - wb) conditions1, 3, 4, many researchers have stated that the wb position is more appropriate for estimating the amount of ankle df motion during functional activities5, 6 . Therefore, wb ankle df prom should be measured during interventions focused on increasing ankle df prom . Limited ankle df prom with knee extended may result from gastrocnemius tightness and insufficient posterior talar glide7 . Thus, gastrocnemius stretching and talocrural joint mobilization have been performed as intervention strategies to increase ankle df prom3, 8, 9 . Previous studies have reported a significant increase in ankle df prom after these interventions3, 8, 9; however, to our knowledge, no study has demonstrated the combined effect of both interventions on wb ankle df prom . Therefore, the aim of the present study was to examine the influence of gastrocnemius stretching combined with joint mobilization on wb ankle df prom . In total, 11 male subjects with bilateral limited non - wb ankle df prom with knee extended (mean age, 22.82 3.09 years; mean height, 175.91 3.39 cm; mean weight, 69.55 3.78 kg; mean non - wb ankle df prom, 4.17 2.48) participated in this study . Inclusion criteria were 1) ankle df prom with knee extension <10; 2) ankle df prom with knee flexion> 10; and 3)> 5 difference in ankle df prom between knee extension and knee flexion conditions on bilateral sides in non - wb positions3 . Subjects with a history of surgery on the lower extremity, fracture, or neurological diseases were excluded from this study . All participants signed an informed consent form approved by the institutional research review committee of inje university prior to participation in this study . The study protocol of this study complies with the ethical standard of the declaration of helsinki . Wb ankle df prom with knee extended was measured following the procedures suggested by munteanu et al10 . Subjects stood in front of a wall and placed the leg being tested behind the contralateral leg in a lunge posture . Subjects were asked to place both hands on the wall and then lean forward without heel - off and knee flexion until maximum stretch was felt in the gastrocnemius on the tested leg . The force applied to the tested leg was maintained at 60 5% of the subject s weight using scales11 . An examiner determined the maximum tibial inclination using an inclinometer to measure the wb ankle df prom with knee extended . Measurements of wb ankle df prom were repeated 3 times for each ankle under pre- and post - intervention conditions . The mean value of 3 trials was used for data analysis . For gastrocnemius stretching combined with joint mobilization, subjects leaned forward against the wall in the same lunge posture as that during measurement of wb ankle df prom with knee extended until the maximum gastrocnemius stretch was felt . Subjects held the end - range posture while an examiner provided the talus of the tested leg with sustained anterior - to - posterior gliding force . An intervention trial was performed for 30 s, and 10 trials were repeated with 30-s rest periods for each ankle . The difference in wb ankle df prom with knee extended between pre- and post - intervention conditions was analyzed using a paired t - test . Wb ankle df prom with knee extended was significantly increased in post - intervention compared with pre - intervention conditions (42.60 5.49 versus 38.24 4.69, p <0.001). Our findings demonstrate that gastrocnemius stretching combined with joint mobilization significantly increases wb ankle df prom with knee extended . Stretching exercises increase tolerance, resulting in increased rom12 . Additionally, increased displacement of the myotendinous junction (mtj) after gastrocnemius stretching for 5 min was found in a previous study13 . Therefore, the change in tolerance and/or increase in mtj displacement might have influenced our findings . The addition of talocrural joint mobilization to gastrocnemius stretching is another possible explanation for our findings . Previous research by dinh et al.3 showed a 4.25 increase in wb ankle df prom with knee extended after gastrocnemius stretching alone for 3 weeks . Although gastrocnemius stretching combined with joint mobilization was applied for 5 min in the present study, the amount of increase in wb ankle df prom after intervention (i.e., 4.36) was similar to that found previously . Considering this outcome, despite the relatively short period of intervention in the present study, it may be inferred that the addition of talocrural joint mobilization might maximize the effects of general gastrocnemius stretching . Thus, we conclude that gastrocnemius stretching combined with joint mobilization might decrease gastrocnemius tightness and increase posterior talar gliding movement, which effectively increases wb ankle df prom with the knee extended . First, although non - wb ankle df prom was used as an inclusion criterion, changes in non - wb ankle df prom after intervention were not measured . However, we believe that wb ankle df prom is clinically more important because most functional activities are performed under the wb condition . Second, our study included only male subjects, and the results cannot be generalized to women.

Dox is one of the key chemotherapeutic drugs for cancer treatment, but its use is limited by chronic and acute toxic side effects . Dox is an antibiotic anthracycline that was isolated from a pigment of streptomyces peucetius in the early 1960s and it had been employed for more than 30 years in the battle against cancer, but it is now chemically synthesized . Renal dox - induced toxicity may be part of a multiorgan damage mediated mainly through free radical formation eventually leading to membrane lipid peroxidation . Induction of apoptosis and modulation of nox are mechanisms that are involved in toxic adverse effects associated with dox therapy . In addition, dox has a direct renal damaging effect as it accumulates preferentially in the kidney . Dox has toxic effects on other organs such as heart and liver which may lead to modulation of blood supply to the kidney and alter xenobiotic detoxification processes, respectively, thus indirectly contributing to dox - induced nephropathy . Dia is a new anti - inflammatory, analgesic, and antipyretic drug that was developed specially for the treatment of osteoarthritis . It is highly effective in relieving the symptoms of osteoarthritis and may be able to modify the course of the disease . Interleukin 1 is a proinflammatory and proapoptotic agent that induces cytokine production by activating nfb and mitogen activated protein kinase signaling . A major cause of dox - induced nephrotoxicity is the production of reactive oxygen species which induce cytokines, including interleukin 1 [6, 9, 10]. The aim of the present study was to study the effect of the interleukin 1 receptor antagonist diacerein (dia) on dox - induced nephropathy . Dia powder was from eva pharma company and it was dissolved in 1% carboxymethylcellulose . Dox hydrochloride 10 mg vial (pharmacia italia, spa, italy), polyclonal rabbit / antirat caspase-3, tnf, and nfb antibody (lab vision, usa), biotinylated goat anti - rabbit secondary antibody (transduction laboratories, usa), urea, gsh, sod, and catalase kits (biodiagnostic, egypt), and creatinine (humen, germany) were purchased . Adult male wistar rats weighing about 250350 g were obtained from the animal research centre, giza, egypt . Animals were kept in standard housing conditions in cages and were left to acclimatize for one week . This work was conducted in the pharmacology department, faculty of medicine, el - minia university, egypt, and the animal experimental protocol was approved by the faculty board . Rats were randomly assigned into 6 groups (n = 6 each) as follows . Group i received vehicle (1% carboxymethylcellulose) for 15 days and ip saline at day 11 . Group ii was treated with dld (25 mg / kg / d orally) for 15 days and ip saline at day 11 . Group iii was treated with dhd (50 mg / kg / d orally) and ip saline at day 11 . Group iv was treated with vehicle for 15 days and dox (15 mg / kg) at day 11 . Group v was treated with dld (25 mg / kg / d orally) for 15 days + ip injection of dox (15 mg / kg) at day 11 . Group vi was treated with dhd (50 mg / kg / d orally) for 15 days + ip injection of dox (15 mg / kg) at day 11 . The doses of dox and dia were based on the previous studies [4, 11]. Group i received vehicle (1% carboxymethylcellulose) for 15 days and ip saline at day 11 . Group ii was treated with dld (25 mg / kg / d orally) for 15 days and ip saline at day 11 . Group iii was treated with dhd (50 mg / kg / d orally) and ip saline at day 11 . Group iv was treated with vehicle for 15 days and dox (15 mg / kg) at day 11 . Group v was treated with dld (25 mg / kg / d orally) for 15 days + ip injection of dox (15 mg / kg) at day 11 . Group vi was treated with dhd (50 mg / kg / d orally) for 15 days + ip injection of dox (15 mg / kg) at day 11 . The doses of dox and dia were based on the previous studies [4, 11]. After 4 days of dox injection, each rat was weighed then anesthetized with ip injection of urethane (25% in a dose of 1.6 gm / kg) and then sacrificed . A longitudinal section of the left kidney and one half was fixed in 10% formalin then embedded in paraffin for histopathological and immunohistochemical examinations . The rest of the kidneys were snap frozen in liquid nitrogen and kept at 80c . Briefly, the method is based on the fact that sulfhydryl group of gsh reacts with 5, 5-dithiobis (2-nitrobenzoic acid) (ellman's reagent) and produces a yellow colored 5-thio-2-nitrobenzoic acid which was measured colorimetrically at 405 nm using beckman du-64 uv / vis spectrophotometer, usa . Assessment of renal catalase antioxidant enzyme activity was determined from the rate of decomposition of h2o2 at 510 nm after the addition of tissue homogenate as described by colorimetric kit . The assessments of sod levels were based on the ability of the enzyme to inhibit the phenazine methosulfate - mediated reduction of nitroblue tetrazolium dye and the results were expressed as unit / g tissue . The renal contents of lipid peroxides were assayed by a spectrophotometric method based on the reaction between mda and thiobarbituric acid . The absorbance values of the samples and the blank were determined at 535 nm using a (beckman du-64 spectrophotometer, usa) and then blank absorbance value was subtracted from the sample absorbance value . From a standard curve, mda concentration in the unknown sample was extrapolated from the corresponding absorbance using the regression line from the standard curve and expressed as nmol / gm tissue by multiplying in the tissue dilution factor . Nitric oxide (no) in the form of nitrite was determined with spectrophotometric method using griess reagent systems . The stable oxidation end products of no, nitrite (no2), and nitrate (no3) were used as indicators of no production . Nox was measured after the reduction of nitrate to nitrite by copperized cadmium granules in glycine buffer at ph 9.7 . Quantification of no2 was based on the griess reaction, in which a chromophore with a strong absorbance at 540 nm is formed by the reaction of nitrite with a mixture of n - naphthylene diamine and sulfanilamide . The absorbance of the sample and the blank were measured at 545 nm using (beckman du-64 spectrophotometer, usa). The blank absorbance is then subtracted from the sample absorbance . From a standard curve, nox content in the unknown sample was extrapolated from the corresponding absorbance using the regression line from the standard curve and expressed as nmol / g tissue . Renal tissue was fixed in 10% formalin, embedded in paraffin, sectioned by a microtome at 5 m thickness and stained with hematoxylin and eosin for routine histopathological assessment . The renal tissues were examined in random microscopic areas semiquantitatively under 40 high power fields and the number of changes was assessed by the counting of 3 nonoverlapped fields for the same slide of each animal . The frequency and the severity of lesions in the kidneys were assessed semiquantitatively as follows: score: assigned normal, score +: in between normal and mild, score + + (mild level): less than 25% of the examined fields revealed histological alterations, score + + + (moderate level): less than 50% of the examined fields revealed histological alterations, and score + + + + (severe level): less than 75% of the total fields examined revealed histological alterations . The caspase-3, tnf, and nfb immunolabeled cells were counted . In each animal, 3 sections were examined and the cells were counted in 3 adjacent nonoverlapping fields levels . Immunohistochemical staining was performed for caspase-3, tnf, and nfb using polyclonal rabbit / antirat antibody according to previously published protocol [20, 21], respectively . Table 1 shows the results of the effect of dia on serum creatinine and urea . Rats receiving a single dose of dox (15 mg / kg, ip) showed a significant increase in serum creatinine and urea levels compared to control group . Both doses of dia resulted in significant decrease in serum creatinine compared to dox treated rats . Dia 50 mg / kg / day but not 25 mg / kg / day resulted in significant decrease in serum urea compared to dox treated rats . Renal mda was evaluated as an indicator of kidney lipid peroxidation and nitrites and nitrates as an indicator of renal nox levels (table 1). Dox (15 mg / kg) significantly increased renal mda and nox levels compared to control group . Administrating both doses of dia to dox treated rats significantly decreased mda and nox compared to dox treated group . Treatment with dox (15 mg / kg) caused significant decrease in renal gsh, sod, and catalase levels compared with untreated control group (table 2). Concomitant treatment of dox with dia significantly increased the levels of renal gsh, sod, and catalase compared to dox treated group . The histological study of the rat renal cortical tissue of control group (figure 1(a)), dld (25 mg / kg / day) group (figure 1(b)), and dhd (50 mg / kg / day) group (figure 1(c)) showed normal architecture of renal glomeruli and tubules . Dox treated group (figure 1(d)) showed marked enlargement of some vascular glomeruli which tightly fill the renal corpuscles . Dox + dld group (figure 1(e)) showed amelioration of the damaging effects of dox . There were less tubular distortion, narrow bowman's spaces, and fewer cytoplasmic vacuolations of renal corpuscle and tubular cells were also observed . Dox + dhd group (figure 1(f)) had more obvious decrease in the morphological changes caused by dox exposure . The severity of the morphological changes was assessed semiquantitatively; dox exposed group showed increase in the glomerular and tubular morphological changes at the light microscopic levels when compared with control group . These changes were suppressed by the administration of both doses of dia, but the high dose showed marked improvement than the low dose (table 3). Administration of dox caused significant increase in the immunoreactivity of caspase-3, nfb, and tnf (figures 2, 3, and 4 and table 4) respectively, which were highly expressed in both renal glomeruli and tubules cytoplasmically and in some nuclei . Administration of both doses of dia concomitantly with dox decreased the expression of them, compared to dox group . Administration of both doses of dia in vehicle treated rats alone and control groups showed no expression . Effective anticancer therapy with anthracyclines as dox is limited because of its toxicity to various organs including kidneys . Nephrotoxic action of dox is also considered to be via drug - induced free radical generation . The formation of free radicals induces the production of proinflammatory cytokines as interleukin 1 initiating the biological effects associated with inflammation . This directed our attention to investigate the role of dia which is interleukin 1 receptor antagonist as a possible nephroprotective agent against dox - induced renal damage . Induction of dox nephrotoxicity was detected in our study by significant elevation of serum urea and creatinine levels which were confirmed by toxic histopathological changes compared to control group . Urea and serum creatinine are the most sensitive markers of nephrotoxicity implicated in the diagnosis of renal injury [24, 25]. The nephrotoxic effect of dox is characterized by decreasing glomerular filtration rate leading to a rise in serum urea and creatinine . Our results are in good agreement with the previous studies [22, 26]. Improvement of dox - induced nephrotoxicity was previously tried by compounds that partially succeeded in preserving normal renal function and structure probably through their antioxidant and anti - inflammatory effects as caffeic acid phenethyl ester, zingiber officinale roscoe, and solanum torvum so that we investigated the role of another antioxidant and anti - inflammatory drug as dia on dox - induced nephrotoxicity . Dia could significantly decrease serum urea and creatinine compared to dox treated group . That is due to the anti - inflammatory and antioxidant effects of dia which suppress dox mediated oxidative stress, inflammation, and tissue damage . Our histopathological changes showed that dox treated group presented with marked damage of renal tubules . These results are in agreement with zhao et al . Who detected the protective effect of rhein (the active metabolite of dia) on acetaminophen induced hepatotoxicity and nephrotoxicity in rats . They found that serum urea and creatinine significantly decreased in rhein and acetaminophen coadministration compared to acetaminophen group and normalization of toxic histopathological changes . The elevated levels of gsh could effectively provide thiol group for the possible gsh mediated detoxification reactions of gpx (glutathione peroxidase) and gst (glutathione - s - transferase) which is involved in the scavenging of o2 generated from the dox . Our findings are consistent with the previous reports that showed that gsh concentration is significantly decreased upon dox treatment compared to control group [4, 22]. Sod extensively distributes in all cells and has a significant shielding role against oxidative injury induced by reactive oxygen species . In our study, the activities of sod and catalase significantly decreased in dox treated rats in kidney as compared to control rats . The accumulation of these highly reactive free radicals leads to the reduction of the activity of sod and catalase which in turn results in damaging effects in the form of loss of cell membrane integrity and function . The decrease in the sod and catalase activities related to the increase in the intracellular levels of h2o2 . Catalase has been reported to be responsible for the detoxification of h2o2, which is an effective inhibitor of sod . Coadministration of dia significantly improved sod, gsh, and catalase levels compared to dox treated group . These results may be due to antioxidant effect of dia which was approved previously by tamura et al . Who indicated the inhibitory effect of dia on indomethacin - induced gastric ulceration which could be mediated by the suppression of reactive oxygen species production based on its inhibition of neutrophil activation and antioxidant activity . In addition, hu et al . Investigated the protective effects of rhein lysinate (rhl), against kidney impairment in senescence - prone inbred strain 10 (samp10) mice . Treatment of samp10 mice with rhl significantly increased the sod and gpx levels in the kidneys . O2 reacts with lipid to form lipid peroxides followed by -oxidation to form mda . That was detected in our study which showed significant increase of mda level in dox treated group compared to control group . These results are in agreement with el - sheikh et al . And yagmurca et al . The high production of nox results in peroxynitrite formation which is a potent and aggressive cellular oxidant and is involved in dox toxicity . The current findings showed that dox administration significantly increased renal level of nox compared to control group and that is in agreement with other studies [26, 37]. These results are in agreement with zhao et al . Who detected the protective effect of rhein on acetaminophen induced nephrotoxicity in rats which was approved by significant decrease of mda and nox on coadministration of rhein plus acetaminophen group compared to acetaminophen group . Our results are in agreement with martel - pelletier and pelletier who reported that no is produced through the activity of inducible nitric oxide synthase and it is a major catabolic factor involved in the pathophysiology of oa . Our results are consistent with hu et al . Who investigated the protective effects of rhein lysinate (rhl), against kidney impairment in senescence - prone inbred strain 10 (samp10) mice . Induction of p53 mediates cell apoptosis through activation of caspase-3 family of proteases and apoptotic cell death . Our study is showing significant increase in caspase-3 expression in dox treated group in comparison with control group . Our study is in consistence with torina et al . Who showed that treatment with dia once a day for 4 weeks after myocardial infarction improved ventricular remodeling by partial blockage of the proinflammatory cytokines which led to lower caspase-3 activity and nfb p65 transcription b pathway . Dox - induced superoxide anion production which was reported to be responsible for tnf-induced nuclear factor (nf) activation that increases nf and tnf over expression . Our study showed significant increase in tnf and nfb expressions in dox group compared to control group and the same results were found with al - saedi et al . . Coadministration of dia significantly decreased tnf and nfb expression compared to dox treated group that is in agreement with gadotti et al . Who showed that dia inhibits neuropathic pain by decreasing proinflammatory cytokines as tnf and nf. Also, hu et al . The active metabolite of dia is rhein which possesses anti - inflammatory activity and may be effective in suppressing the inflammatory cytokines contributing to the pathogenesis of diabetic nephropathy . . Demonstrated that rhein had protective effect in different models of nephropathy as iga induced nephropathy, obstructive nephropathy, chronic allograft nephropathy, and high glucose and angiotensin ii induced nephropathy . Oral administration of rhein (150 mg / kg / d) ameliorated renal lesions . Rhein was capable of protecting against renal injury by decreasing the activities of nfb and caspase-3 in the early phase of glomerulosclerosis . Our results are consistent with meng et al . Who reported that rhein possesses various pharmacological activities, including anti - inflammatory, antioxidant, and antitumor . In their study, a model of hyperuricemia and nephropathy induced by adenine and ethambutol in mice was established . The results demonstrated that rhein significantly improved the symptoms of nephropathy through decreasing the production of proinflammatory cytokines, including interleukin 1, prostaglandin e2, and tnf. Yu et al . Aimed to explore the effect of rhein on sepsis - induced acute kidney injury by injecting lipopolysaccharide (lps) and cecal ligation and puncture (clp) in vivo and on lps - induced hk-2 cells in vitro . Rhein could significantly decrease concentration of serum urea and creatinine and level of tnf, nfb, and il-1 in two different mouse models of experimental sepsis . In conclusion, dia protected against dox - induced nephrotoxicity in rats most probably due to its antioxidant and anti - inflammatory activities . However, dhd (50 mg / kg / day) showed more protective effect than dld (25 mg / kg / day).

Suicide is a leading cause of death among adolescents and has become a significant public health issue globally (1). Statistics korea report 2014 showed that suicide is the cause of 28.4% of adolescent deaths in 2013, and it was the number one cause of death among adolescents (2). Furthermore, suicide is the second to third cause of mortality among adolescents in the western world and is an important concern in developing countries (3). Numerous causes of suicidal behavior during adolescence have been reported, including mental disorders, peer affiliations, and level of achievement in school (4). Additionally, weight - related concerns are a common cause of suicidal behaviors (56). This transition often leads to confusion about personal identity, especially in the context of enduring developments in identity and cognition as well as physical growth (7). Adolescents typically enter a period of a high level of self - consciousness about their body shape and size (8). They have a greater tendency to consider how they look to others at this life stage than during any other period of life (9). These weight - related issues are influenced by many factors including friends, peers, social norms, and media (10). The media is a particularly strong influence because it has become a general source of information and adolescents are easily exposed to it (11). The media encourages as " ideal " slim and thin body shapes for girls and muscular bodies for boys (1213), and provides abundant information on the newest diet methods and quick fixes for weight control (14). This may induce adolescents, who have a particularly keen interest in their figure, to distort their body image and to engage in unneeded or inappropriate weight control behaviors (wcb) (15). Many adolescents engage in inappropriate wcb such as vomiting, taking nonprescription diet pills, and taking laxatives or diuretics . Among korean adolescents engaging in wcb, 13.4% of boys and 18.8% of girls took inappropriate methods to control their weight (16). According to a study using data from national surveys, similar rates were also observed in the united states, with 10% of boys and 21.3% of girls conducting at least one inappropriate wcb (17). Inappropriate wcb are a harmful influence on adolescents' physical and psychological development (12). For instance, a 5-year longitudinal study reported that engagement in inappropriate wcb can be a predictor of obesity and eating disorders (18). Moreover, adverse outcomes of inappropriate wcb also include the onset of depression (19) and suicide ideation (20). Due to the high prevalence of and the possible negative side effects associated with inappropriate wcb, it is a growing serious public health concern . Therefore, the prevention of inappropriate wcb among adolescents is imperative to protect them from negative physical and psychosocial consequences . To date, studies about inappropriate wcb merely suggest the association between inappropriate wcb and negative psychosocial consequences (212223). In the present study, we investigate not only the association between inappropriate wcb and suicide ideation and attempt, but also the difference in this association with body mass index, body weight perception, and body shape misperception . Moreover, we investigate the top five wcb combinations used by korean adolescents and examine the association between these five wcb combinations and suicide ideation and attempt . We used a cross - sectional design; thus, the results should be interpreted with caution due to possibility of a bi - directional effect . We used data from the 2014 korea youth risk behavior web - based survey (kyrbs). The kyrbs has been conducted annually since 2005 by the korean centers for disease control and prevention (kcdc), the korea ministry of education, science and technology, and the korea health and human services . The data were collected via an ongoing, anonymous web - based survey in a self - reporting format that was conducted on a nationally representative sample of middle- and high - school students . It aimed to plan and assess the korean adolescent health promotion policies by investigating health - related behaviors and status . In the 2014 survey, 799 middle and high schools were selected, including 72,060 students in grades 7 to 12 (stratified) on a national scale . However, we excluded 2,334 individuals due to missing data for variables used in this study; therefore, our cohort ultimately comprised 69,726 adolescents (35,224 boys and 34,361 girls). Suicide ideation and attempt were measured via responses to the following questions: " have you seriously considered suicide during the past 12 months? " And " have you tried suicide during the past 12 months? " The possible responses to both questions were " yes " or " no . " We characterized wcb via responses to the following multi - part question: " have you experienced the following weight control methods during the past 30 days, with the following activities listed: 1) did regular exercise, 2) fasted at least 24 hours, 3) ate less, 4) took prescription diet pills, 5) took nonprescription diet pills, 6) took laxatives or diuretics, 7) vomited, 8) ate only one food, 9) took oriental medicine, and 10) ate diet food . " The possible responses to all questions were " yes " or " no . " If participants responded' yes' to at least one of 2), 5), 6), 7), and 8), we classified them into the " inappropriate wcb " group (16). If participants responded' no' at all of 2), 5)-8), we classified them into the " appropriate wcb " group . If participants responded' no' to all activity choices, we classified them into the " nothing " group . Body mass index (bmi) was calculated by self - reported height and weight . Bmi percentiles for age and gender were calculated according to the 2007 standard growth charts for korean children and adolescents by the korean pediatric society (24), and four categories were created: underweight (15th percentile), normal weight (16th to 84th percentile), overweight (85th to 95th percentile), and obese (95th percentile or bmi 25). We further grouped overweight and obese groups into a single overweight group, thus using three bmi categories body weight perception (bwp) was split into five categories: very underweight, underweight, normal weight, overweight, and very overweight . We further grouped these categories into three groups: thin, normal, and obese . Body shape misperception (bsm) was determined by an agreement between bmi and bwp categories . Participants perceiving their weight at least one category below their actual bmi categories were designated part of the " underestimate " group, while those perceiving their weight at least one category above their actual bmi were designated part of the " overestimate " group (i.e., if participants whose bmi was a normal weight perceived their body weight as obese, they were placed in the " overestimate " group) (25). Control variables included age, household economic status, parents' presence, residential area, school level, academic achievement in school, subjective health status, sleep satisfaction, physical activity, current alcohol consumption, current smoker, stress level, and depression . We performed statistical analyses of the survey data using sas version 9.4 (sas inc ., the relevant primary sampling units, sample weights, and stratification were considered in the analysis given that the kyrbs was designed as a complex sample . A pearson's test was used to determine significant differences in the distribution of each variable . Next, a logistic regression analysis was conducted to determine the association between wcb and suicide ideation and attempt . Additionally, we performed a set of subgroup analyses using bwp, bmi, and bsm to determine whether body related variables led to differences in the association between wcb and suicide ideation and attempt . Results were presented as adjusted odds ratios (ors) with 95% confidence intervals (95% cis). The ethics approval for the publicly open kyrbs data was waived by the institutional review board (irb). After the purpose of the survey was fully explained to the students, written informed consent was provided . Students were assured that they could refuse or withdraw from the research at any stage (16). We used data from the 2014 korea youth risk behavior web - based survey (kyrbs). The kyrbs has been conducted annually since 2005 by the korean centers for disease control and prevention (kcdc), the korea ministry of education, science and technology, and the korea health and human services . The data were collected via an ongoing, anonymous web - based survey in a self - reporting format that was conducted on a nationally representative sample of middle- and high - school students . It aimed to plan and assess the korean adolescent health promotion policies by investigating health - related behaviors and status . In the 2014 survey, 799 middle and high schools were selected, including 72,060 students in grades 7 to 12 (stratified) on a national scale . However, we excluded 2,334 individuals due to missing data for variables used in this study; therefore, our cohort ultimately comprised 69,726 adolescents (35,224 boys and 34,361 girls). Suicide ideation and attempt were measured via responses to the following questions: " have you seriously considered suicide during the past 12 months? " And " have you tried suicide during the past 12 months? " The possible responses to both questions were " yes " or " no . " We characterized wcb via responses to the following multi - part question: " have you experienced the following weight control methods during the past 30 days, with the following activities listed: 1) did regular exercise, 2) fasted at least 24 hours, 3) ate less, 4) took prescription diet pills, 5) took nonprescription diet pills, 6) took laxatives or diuretics, 7) vomited, 8) ate only one food, 9) took oriental medicine, and 10) ate diet food . " If participants responded' yes' to at least one of 2), 5), 6), 7), and 8), we classified them into the " inappropriate wcb " group (16). If participants responded' no' at all of 2), 5)-8), we classified them into the " appropriate wcb " group . If participants responded' no' to all activity choices, we classified them into the " nothing " group . Body mass index (bmi) was calculated by self - reported height and weight . Bmi percentiles for age and gender were calculated according to the 2007 standard growth charts for korean children and adolescents by the korean pediatric society (24), and four categories were created: underweight (15th percentile), normal weight (16th to 84th percentile), overweight (85th to 95th percentile), and obese (95th percentile or bmi 25). We further grouped overweight and obese groups into a single overweight group, thus using three bmi categories body weight perception (bwp) was split into five categories: very underweight, underweight, normal weight, overweight, and very overweight . We further grouped these categories into three groups: thin, normal, and obese . Body shape misperception (bsm) was determined by an agreement between bmi and bwp categories . Participants perceiving their weight at least one category below their actual bmi categories were designated part of the " underestimate " group, while those perceiving their weight at least one category above their actual bmi were designated part of the " overestimate " group (i.e., if participants whose bmi was a normal weight perceived their body weight as obese, they were placed in the " overestimate " group) (25). Control variables included age, household economic status, parents' presence, residential area, school level, academic achievement in school, subjective health status, sleep satisfaction, physical activity, current alcohol consumption, current smoker, stress level, and depression . We performed statistical analyses of the survey data using sas version 9.4 (sas inc ., the relevant primary sampling units, sample weights, and stratification were considered in the analysis given that the kyrbs was designed as a complex sample . A pearson's test was used to determine significant differences in the distribution of each variable . Next, a logistic regression analysis was conducted to determine the association between wcb and suicide ideation and attempt . Additionally, we performed a set of subgroup analyses using bwp, bmi, and bsm to determine whether body related variables led to differences in the association between wcb and suicide ideation and attempt . Results were presented as adjusted odds ratios (ors) with 95% confidence intervals (95% cis). The ethics approval for the publicly open kyrbs data was waived by the institutional review board (irb). After the purpose of the survey students were assured that they could refuse or withdraw from the research at any stage (16). The demographics of the 35,224 boys and 34,361 girls in this study are listed in table 1 . Of the participants, 10.6% (n = 3,709) of boys and 15.0% (n = 5,172) of girls reported that they experienced suicide ideation, while 2.1% (n = 722) of boys and 3.4% (n = 1,151) of girls reported attempting suicide . 33.5% (n = 11,827) of boys and 45.2% (n = 15,489) of girls reported that they engaged in appropriate wcb, and 4.2% of boys (n = 1,466) and 9.6% of girls (n = 3,324) reported that they engaged in inappropriate wcb . Boys who had engaged in inappropriate wcb were more likely to report suicide ideation than those not engaging in wcb group (or, 1.57, p <0.001). Girls engaging in inappropriate wcb also had a high suicide ideation rate (or, 1.32, p <0.001). Boys engaging in inappropriate wcb were also more likely to attempt suicide than those not engaging in wcb (or, 2.49, p <0.001). Girls engaging in appropriate wcb (or, 1.21, p = 0.02) or inappropriate wcb (or, 1.92, p <0.001) were more likely to attempt suicide than girls not engaging in wcb at all, and the association was stronger among the latter comparison group . The association between suicidal behavior and wcb stratified by bmi, bwp, and bsm is shown in table 3 . Boys with an underweight bmi and that engaged in inappropriate wcb experienced the highest rate of suicide ideation (or, 2.22, p <0.05) compared to other bmi groups . Girls with an underweight bmi and that engaged in inappropriate wcb were 4.53 times more likely to attempt suicide than girls who did not engage in wcb . Girls who perceived their weight as normal and engaged in inappropriate wcb were more likely to experience suicide ideation (or, 1.50, p <0.001) and attempt (or, 2.31, p <0.001) than girls who did not engage in wcb . Considering bsm, boys who distorted their body shape and who engaged in inappropriate wcb were more likely to experience suicide ideation (underestimate: or, 1.44, p <0.01, overestimate: or, 1.91, p <0.01) and attempt (underestimate: or, 1.96, p <0.01, overestimate: or, 2.94, p <0.01) than boys who do not engaged in wcb . Girls who underestimated or overestimated their body shape and engaged in inappropriate wcb showed significant association with suicide attempt (underestimate: or, 1.89, p <0.001, overestimate: or, 1.73, p <0.001) compared to girls who do not engaged in wcb . Adjusted for age, sex, household economic status, parents presence, residential area, school level, academic achievement, subjective health status, sleep satisfaction, physical activity, current alcohol consumption, current smoker, stress status, and depression . Cannot be calculated due to the lack of number . Table 4 lists the odds ratios of the top five wcb combinations engaged in by korean adolescents and their association with suicide behaviors . Conducting regular exercise and eating less together with fasting was significantly associated with suicidal ideation (boys: or, 1.70, p <0.01, girls: or, 1.33, p <0.05) and attempt (boys: or, 1.84, p <0.05, girls: or, 2.24, p <0.001) among both boys and girls, and girls had higher rates of attempted suicide . Adjusted for age, sex, household economic status, parents presence, residential area, school level, academic achievement, subjective health status, sleep satisfaction, physical activity, current alcohol consumption, current smoker, stress status, and depression . In the present study, we investigated association between inappropriate wcb and suicide ideation and attempt by korean adolescent by using nationally represented data . Inappropriate wcb was significantly associated with suicidal behaviors in both boys and girls, and with a higher rate in boys . Our study findings of a higher likelihood of suicidal behaviors among adolescents engaging in wcb (especially inappropriate wcb), confirm results described in previous studies . Adolescents using inappropriate methods to control weight are highly likely to experience psychological symptoms including anxiety, fatigue, and impaired concentration (26). Additionally, there is a prospective association between wcb and depression (27). Depression in adolescence is a serious public health concern due to the increased risk of suicide among depressed youth (28). Moreover, wcb can cause eating disorders, with fasting an especially high risk factor for development of eating disorders (29). Another study among adolescents and with a 5 year follow up reported that unhealthy wcb were the most powerful predictors of eating disorders (18). Eating disorders have adverse consequences on adolescents' physical and mental health (30), and can cause suicide ideation and attempt (31). Therefore, one possible interpretation of this finding is that wcb represent a risk factor for suicide ideation and attempt by causing depression or eating disorders, both of which have serious consequences on adolescents' mental health . Furthermore, our study demonstrated stronger associations among boys than girls, which is corroborated by other studies (623). Girls are more sensitive to their weight and easily engage in inappropriate wcb (32), whereas boys are not as sensitive about their weight and have a lower likelihood of engaging in inappropriate wcb (7). Therefore, it is possible that boys who consider controlling their weight in inappropriate ways may already be suffering from severe psychological distress due to experiencing immense trauma from teasing or other adverse events . We investigated differences in association between suicide ideation and attempt and wcb by conducting subgroup analyses using bmi, bwp, and bsm . In the context of bmi, underweight boys engaged in inappropriate wcb experienced the highest rate of suicide ideation and attempt than other bmi groups, and underweight girls were considerably more likely to attempt suicide . Additionally, suicidal behavior was also associated with boys and girls of a normal weight engaged in inappropriate wcb . This suggests that adolescents who do not need to control their weight but engage in unnecessary wcb nonetheless are at higher risk for experienced adverse consequences (12). With regards to bsm, the results of this study showed that misperception of body shape is also associated with suicide ideation and attempt . The media continues to portray slender bodies for girls and muscular bodies for boys as ideal body shapes, and this could have an effect on body shape misperception among adolescents (33). This mismatch contributes to adolescent depression, which in turn increases the risk of suicide among youth (34). Moreover, this mismatch could be related to adolescents' unnecessary weight control behaviors to obtain the ideal body shape . Eventually, the effect of inappropriate wcb aggravates adverse effects from a distorted body image, and it seems that this psychological burden causes adolescents to think about or attempt suicide . We also extracted the five most common combinations of weight control methods used by korean adolescents and investigated the association between these wcb and suicidal behaviors . Only conducting regular exercise was the most frequent wcb among boys and the third most common among girls . However, this combination was not significantly associated with suicidal behaviors in boys or girls . The most common wcb engaged in by girls was' regular exercise and eating less', and it also did not show any significant association with suicidal behaviors . Interestingly, both boys and girls engaging in' regular exercise, fasting, and eating less' were more likely to experience suicidal behaviors compared to those who did not conduct any wcb . Although regular exercise and eating less are both appropriate wcbs, when fasting, which is classified as an inappropriate wcb, is added, this combination could contribute to adolescents' suicidal behaviors . According to a previous study, fasting for weight control showed an association with depression when compared with other wcbs (35). In practice, fasting causes the blood glucose concentration to fall below normal, and this status releases the hormone cortisol, which is associated with anxiety and negative feelings (36). Therefore, it seems that this combination influences suicidal behaviors due to the strong influence of fasting on depressive symptoms . This study was designed as cross - sectional study; therefore, we could not exclude the potential bi - directional effect . In addition, the time frames of wcb (within the past 30 days) and suicide ideation and attempt (within the past 12 months) differed . Therefore, a reverse - time order could not be clearly excluded (6). Moreover, responses from the kyrbs represent self - reported data, and therefore may be inaccurate . Additionally, we could not obtain detailed descriptions of each wcb including frequency or amount . However, despite the limitations, this study can be generalized by using nationally representative data . Additionally, compared to previous studies that only reported associations between wcb and suicidal behavior, we further investigated differences in this association in the context of bmi, bwp, and bsm . Moreover, this is the first study to investigate common wcb combinations and to determine the association between the top five such combinations and suicide ideation and attempt . Considering that the high incidence of suicide and the severe negative impact of adolescents' inappropriate wcb on their health, our findings represent an important motivation for health policy makers to identify solutions for controlling adolescents' suicide problem . Inappropriate wcb among adolescents is a serious public health concern considering its widespread prevalence and harmful influence on growth, physical health, and psychosocial growth . Additionally, as shown here, wcb can be associated with health compromising behaviors including suicide ideation and attempt . Therefore, to protect adolescents' healthy psychological status in the context of weight concerns, policy makers and health professionals must endeavor to correct the distorted body perception experienced by adolescents . Additionally, they should help adolescents develop skills for avoiding inappropriate wcb, and encourage appropriate methods of weight control such as physical activity, fruit and vegetable consumption, and the avoidance of fatty and sweet foods . Future studies should be designed regardless of gender or weight status because boys and normal or underweight adolescents are currently low priority even though they are also at risk for adverse effects due to wcb according to the present study . Therefore, broader viewpoints and approaches are needed when considering adolescents' weight - related behaviors.

In the past 15 years, green fluorescent protein (gfp) has changed from a nearly unknown protein to a commonly used molecular imaging tool in biology, chemistry, genetics, and medicine . In 2006, more than 10 000 papers gfps and gfp - like proteins (i.e., chromoproteins and fluorescent proteins) are particularly useful due to their stability and the fact that the chromophore (see figure 1) is formed in an autocatalytic cyclization of the 65syg67 sequence that does not require a cofactor . This means that unlike most other bioluminescent reporters, gfp fluoresces in the absence of any other proteins, substrates, or cofactors . Furthermore, it appears that fusion of gfp to a protein does not alter the function or location of the protein . By changing residue 66 and/or the amino acid residues around the chromophore (n1c1c2c3) and (c1c2c3c4) dihedral angles of the gfp chromophore . In the protein, r1 is gly67 and r2 is ser65, and in hbdi, an often - used model compound, r1 = r2 = ch3 . In one - bond flips (-obf) the dihedral rotation occurs around the torsional angle, in a -obf it is around the dihedral angle, and in a positively correlated hula - twist (+ ht) the and dihedral angles concertedly rotate in the same direction (as shown above), while in a negatively correlated hula twist (ht) they concertedly rotate in opposite directions . A plot of the and dihedrals for a perfectly correlated negative ht will have a slope of 1 . If the chromophore cavity is complementary with a planar chromophore, then the and best fit line will pass through the origin and all the and angles will be centered around the origin . A nonzero intercept along the or axis (see, for example, figure 4) or and dihedrals centered in quadrant ii (> 0; <0) or quadrant iv (<0;> 0; see, for example, figure 8) are indications of a cavity that is not complementary with a planar chromophore . The fluorescent emission of the chromophore within gfp occurs with high efficiency (quantum yield fl = 0.8) and a respectable fluorescence lifetime (3 ns). (19) when the protein is denatured the fluorescence yield decreases by at least 3 orders of magnitude. (20) model compounds of the chromophore do not fluoresce in solution (quantum yield fl <10), unless the rotation of the arylalkene bond is restrained. (21) fluorescence can, however, be obtained by lowering the temperature to 77 k; this freezes the solution . It has been suggested that twisting between the phenolate and imadazolidinone groups of the chromophore is the mechanism for the ultrafast fluorescence quenching internal conversion process . Neutral form (a) of the chromophore, with the phenolic oxygen protonated, can convert to the anionic species (b) by going through an intermediate state (i). The change from forms a to i is solely a protonation change, while the change from i to b is a conformational change with most changes occurring at thr203 . Upon excitation of the a state, an excited - state proton transfer (espt) occurs in which the proton is transferred from the chromophore to glu222 in a time scale of the order of picoseconds . Following radiative relaxation from the excited - state intermediate (i *), the systems returns to the ground state a through the ground - state intermediates i1 and i2. (29) excitation of the anionic b state results in direct emission from the excited state (b *) at 482 nm . Recently, a nonfluorescent dark state, state c, has been observed that is distinct from states a and b and absorbs at higher energies. (30) the c state, perhaps the neutral trans form of the chromophore, may be populated by nonradiative decay from a * and it may be depopulated by excitation to the excited c * state with transcis isomerization to repopulate state a. fluorescent blinking has been ascribed to nonadiabatic crossing and conversions between the neutral and anionic states,(31) or a possible dark (z) zwitterionic state . While the ground - state minimum of the gfp chromophore is close to planar, this is not necessarily so for the excited state; in fact, in some cases the excited state has an energy minimum with a twisted chromophore in which both rings are 90 to each other. (32) according to quantum mechanical calculations, the ground and excited states for the one - bond flip (obf) and hula twist (ht) in the neutral form (a) and the obf in the zwitterionic form come very close to each other . It has been proposed that in the absence of the protein matrix, which surrounds the chromophore and prevents twisting, this process can lead to fluorescence quenching internal crossing;(32) see figure 3 . Recent calculations on the gfp chromophore model compound hbdi suggest that the anionic form of hbdi may also undergo a -obf that leads to a favored radiationless decay channel, which is particularly efficient in solvent. (34) (a) model compounds of the gfp chromophore in the ground state (s0) can be excited to the first singlet state (s1) in which a ht or obf can freely occur . Upon reaching the perpendicularly twisted conformation, fluorescence quenching nac (nonadiabatic crossing) occurs . (b) in the ground state (s0), the residues surrounding the gfp chromophore exert a twisting force on the chromophore (). Upon excitation, the conjugation across the ethylenic bridge of the chromophore is reduced and it will twist; however, the protein matrix prevents the chromophore from reaching the perpendicularly twisted conformation () and fluorescence quenching internal crossing is prevented . Recently, the results from a number of interesting experiments that provide evidence for chromophore twisting and/or fluorescence quenching internal crossing (ic) have been published . For example, a molecular dynamics simulation of the chromophore of cyan fluorescent protein found that the average dihedral angle was about 0; however, when the surrounding protein was considered in the simulation the average shifted by approximately 5, showing that the protein matrix of cfp twists the chromophore . On the basis of their calculations, the authors concluded that the driving force for this twist comes from the strong short - range repulsion by four residues (ile167, val150, phe165, and thr203) surrounding the average position of the chromophore. (46) photoswitching fluorescent proteins can be switched back and forth between the naturally occurring green state and a dark state by 405 nm irradiation (e.g., dronpa, mtfp0.7, kfp1). A cistrans isomerization of the chromophore has been proposed as the structural basis for the photoswitching observed in dronpa. (47) this is supported by the fact that mutating either val157 or met159 with smaller residues accelerates photoswitching, presumably by decreasing steric hindrance to cistrans isomerization. (48) the m159 t and v157 g mutations also decrease the quantum yield of dronpa from 0.85 to quantum yields of 0.23 and 0.77, respectively. (48) recently, it has been suggested that adoption of a trans configuration cannot solely be responsible for the nonfluorescent form. (49) on the basis of nmr analyses, miyawaki et al . Propose that the fluorescence of the protein is regulated by the degree of flexibility of the chromophore but is not necessarily accompanied by cistrans isomerization. (49) interestingly, gfp is not unique in photoactive yellow protein (pyp) the protein matrix also prevents the chromophore from adopting a completely planar structure . In pyp the asymmetric proteinchromophore interaction probably serves as the initial accelerant for the light induced photocycle,(50) which ultimately leads to a cistrans isomerization. (51) the chromophore in wild - type gfp is planar due its extended system (see figure 1); however, the energy barrier to deformation is low and the protein matrix in wild - type gfp exerts some strain away from planarity . When the chromophore is computationally permitted to freely rotate, it will adopt a conformation that complements the protein matrix . Recently, we have used computational methods to show that wild - type gfp is not an anomaly and that all gfp and gfp - like proteins in the protein databank have a protein matrix that is not complementary with a planar chromophore. (54) in most cases the freely rotating chromophore undergoes rotations of at least 20. in some cases these rotations are accompanied by an equal but opposite rotation of the dihedral angle (a negatively correlated ht). None of the proteins examined have a cavity that causes a rotation solely around the dihedral angle. (54) these calculations were done by minimizing, with freely rotating and dihedral angles, the crystal structure of 38 gfp analogues and mutants found in the pdb . They found the energy minimum conformation of a freely rotating chromophore in the protein matrix of the gfp mutant or gfp - like protein examined; however, they did not provide any information about the range of low - energy conformations available to a freely rotating chromophore . To get this information, we ran molecular dynamics simulations of some of the interesting gfp mutants in the protein databank. (56) by running molecular dynamics simulations, with freely rotating and dihedrals, we have been able to determine the range of conformations available to chromophores with complete rotational freedom . The coordinates of three crystal structures (1gfl,(57) 1myw,(58) 2emd(59)) were obtained from the protein data bank (pdb),(56) and hydrogen atoms were added to protein and solvent atoms as required . The opls_2005 force field of macromodel v9.0016(60) was used . Starting structures for mutants for which no crystal structure has been determined were calculated by graphically mutating a known structure and undertaking a conformational search . The flexible dihedral angles of all the side chains of residues that are within 8.00 of the chromophore were randomly rotated by between 0 and 180, and all solvent molecules in that sphere were randomly rotated and translated by between 0 and 1.00 in each monte carlo (mc) step. (63) 15 000 mc steps were taken in each search . Structures within 50 kj / mol of the lowest energy minimum were kept and a usage - directed method(62) was used to select structures for subsequent mc steps . Structures found in the conformational search were considered unique if the least - squared superimposition of equivalent non - hydrogen atoms found one or more pairs separated by 0.25 or more . The lowest energy structure obtained in the monte carlo torsional and molecular position variation search the final structures obtained from the mc search (monte carlo torsional and molecular position variation and large - scale low mode) or fully minimized pdb structures were used to initiate md simulations with freely rotating and dihedral angles (v1 = v2 = v3 = 0.000). The predynamics simulation was set for 100 ps and the full md simulation for 5000 ps . The cambridge structure database (csd) v5.29 was released in january 2008; it comprises 436 384 small molecule crystal structures . The area of the convex hull of the and graphs was calculated as was the smallest convex set containing the region . Molecular mechanics and dynamics calculations have been used to examine the steric environment of the chromophore in gfp in its ground state . They are techniques that are based on classical physics and were designed to model structural and not electronic properties; therefore, molecular mechanics and dynamics simulations of gfp cannot examine the excited state of the chromophore . Quantum calculations are an excellent technique to determine the energy profiles of the ground and excited states of the chromophore; however, they are cpu - intensive and it would be cost prohibitive to do conformational searches on the excited state of a series of gfp and gfp - like proteins . Therefore, in an attempt to supplement the quantum calculations, we have examined the conformational space available to the chromophore within gfp using a freely rotating chromophore that is an approximation (based on qm calculations) of the conformational space available to the chromophore in its excited state . As shown in figure 2, gfp adopts two states, the neutral state (a) of the chromophore with the phenolic oxygen protonated, and the anionic species (b) the change from forms a to i is solely a protonation change, while the change from i to b is a conformational change with most changes occurring at thr203. (67) in order to see whether the gfp chromophore has different dihedral freedom in forms a, b, and i, we conducted molecular dynamical simulations with a freely rotating chromophore, as described in the . Not surprisingly, table 1 and figure 4 show that forms a, b, and i of gfp have similar dihedral freedom and that all undergo similar negatively correlated (same slopes of best fit lines in figure 4) hula twists . Plot of the vs dihedral angles (see figure 1 for nomenclature) for the 2000 gfp - a (pink), gfp - b (green), and gfp - i (light - blue) structures obtained in the freely rotating molecular dynamics simulation . However, there are subtle differences; forms a and i are closer to the planar ground - state conformation, while the b form adopts conformations further from planarity when given rotational freedom around the and dihedrals (intercept further from = 0); and the anionic b form has the most freedom (largest area, and and range), while the neutral a form has the least freedom . The a form has robust hydrogen bonds between both arg96 and gln94 and the imidazolidinone carbonyl group that remain intact throughout the simulations . In the intermediate (i) form these hydrogen bonds are supplemented by an additional hydrogen bond between the phenolic oxygen of the chromophore and his148 (see table 2). The main difference between the anionic b form and the intermediate form is that thr203 has to rotate in order to hydrogen bond to the phenolic oxygen in form i, otherwise they have the same hydrogen - bonding interactions throughout the simulation . The y66h mutant of gfp exhibits blue fluorescence and has therefore been termed blue fluorescent protein . The crystal structure of bfp (not to be confused with blue fluorescent protein from aequorin bfp - aq(68)) has been solved at ph 4.5(27) and ph 8.5;(59) the overall fold of the protein is identical to wild - type gfp and consists of a chromophore surrounded by an 11-stranded -barrel . While gfp has absorption maxima at 395 and 475 nm and emits at 508 nm, bfp absorbs at 382 nm and emits at 448 nm. (9) unfolding of bfp results in an absorption red shift of 15 nm,(27) and quantum mechanical calculations suggest that the 15 nm shift might be due to the protein - induced nonplanarity of the chromophore. (69) blue fluorescent protein (bfp) has a much lower fluorescence quantum yield than gfp (fl = 0.20 vs 0.80). It has been suggested that this is due to the fact that his66 (bfp - chromophore) forms fewer hydrogen bonds with the surrounding protein than tyr66 (gfp - chromophore) does, and that the smaller imadazole ring (his66) in bfp may have more conformational freedom than the larger phenol (tyr66), which leads to more intersystem crossing. (27) in a very elegant series of experiments, boxer et al . Have shown that the fluorescence quantum yield of blue fluorescent protein increases from fl = 0.20 to 0.35 when the pressure is increased from atmospheric pressure to 570 mpa. (70) analysis of the fluorescence lifetimes in the picosecond and nanosecond regimes reveals that the enhancement of the fluorescence quantum yield is due to the inhibition of fast quenching processes . Temperature - dependent fluorescence measurements reveal two barriers (19 and 3 kj / mol, respectively) for the transition into nonfluorescing states . These steps are probably linked with dissociation of the hydrogen bond between the chromophore and his148 or an intervening water molecule and to the barrier for chromophore twisting in the excited state, respectively. (70) in order to establish the consequences of the y66h mutation on the flexibility of the chromophore, molecular dynamics simulations of the ph 8.5 bfp crystal structure (pdb code 2emd with neutral imidazole rings for his66 and his148) with a freely rotating chromophore were run . [we thank one of the referees for suggesting the ph 8.5 (2emd) over the ph 4.5 (1bfp) structure .] Figure 5 and table 1 show that the chromophore in bfp has significantly more rotational freedom than that available to the chromophore in gfp, and that the bfp cavity is less complementary to a planar chromophore than the gfp cavity is; that is, the bfp sampled structures are further from a planar chromophore (= = 0) and the intercept for the best fit line through the bfp structures is further from the origin than that of gfp . Plot of the vs dihedral angles for the 2000 bfp (blue) and gfp - a (green) structures obtained in the freely rotating molecular dynamics simulation . Analysis of the 2000 bfp structures revealed a hydrogen bond between the nh of the his66 imidazole ring and glu222, which is retained through - out the entire simulation (see figure 7). The smaller imadazole ring of the bfp chromophore results in more dihedral freedom for the chromophore in bfp than phenol in gfp; however, there seems to be no large difference in the number and stability of hydrogen bonds formed by the chromophore in the two fps . Recently, two new blue fluorescent proteins (azurite and a5) with enhanced brightness and photostability were created . The methodology applied to find the brighter bfp mutants was based on the concept that replacing the residues surrounding the chromophore with bulkier amino acids would constrain the chromophore s motion and thereby increase the proteins brightness . The crystal structure of a5 has yet to be solved . In order to find a starting conformation for the md simulation, the 2emd crystal structure was graphically mutated to a5 and a thorough conformational search was undertaken (see). Given the high structural similarity between all the solid state structures of gfp mutants in the protein databank, a thorough conformational search should find the lowest energy conformations of a5 by making the relevant mutations to the 2emd structure . Figure 6 and table 1 show that the conformational space available to the chromophore in bfp is larger than that in a5 and that the bfp conformations tend to be further from planarity; i.e., for a5 median = 2.92, median = 2.22 and intercept of best - fit line is 4.62 vs for bfp median = 27.59, median = 10.18 and intercept of best - fit line 14.27 . Similar results were found for md simulations carried out from all of the five lowest energy conformational families sampled in the conformational search of a5 and in a md simulation initiated from an a5 structure obtained by taking the 2emd structure, graphically mutating it to a5, and thoroughly minimizing the structure without undertaking a conformational search . Plot of the vs dihedral angles for the 2000 bfp (dark blue) and a5 (pink) structures obtained in the freely rotating molecular dynamics simulation . The n of the his66 imidazole ring hydrogen bonds with water305 (2emd numbering) in all the sampled structures . This water connects his66 to the surface of the protein via a robust hydrogen - bonding network to his148 and is also hydrogen bonded to glu222 . In half the structures sampled, the n of the his66 imidazole ring hydrogen bonds to water302 which is in an extensive hydrogen - bonding network with val68, arg224, and gln69 . In contrast to 2emd there are no hydrogen - bonding interactions with the n hydrogen; see figure 7 . Hydrogen - bonding interactions (---) for 2emd (top) and a5 (bottom) chromophore . These results seem to indicate that the enhanced brightness of a5 is at least in part due to the fact that its chromophore movement is restricted relative to bfp . This restriction can be due to both steric factors and the increased hydrogen - bonding networks in a5 . In the first rationally designed mutant based on the crystal structure of gfp - s65 t, it was decided to mutate t203 into a tyrosine so that it could stack with the phenolic group in the chromophore . (73) the resultant mutant, yfp, is red - shifted by 16 nm relative to gfp - s65 t and does indeed have a stacking interaction between the chromophore and tyr203. (74) on the basis of the crystal structure of yfp, remington et al . Proposed that the red shift was due to the additional polarizability of the -stacked tyr203, the hydrogen - bond pattern around the chromophore, or an out - of - plane distortion of the chromophore (in analogy with out - of - plane distortions in porphyrin systems). (74) comparison with the t203v mutant revealed that the t203y substitution leads to a significant i - form population and only about 10 nm of the shift can be ascribed to the interaction. (75) a new varient of yfp, venus, with improved brightness and maturation properties as well as a reduced environmental sensitivity was developed and crystallized. (58) since some researchers have argued that the presence of tyr203 leads to a decrease in chromophore flexibility (van der waals volume for tyr = 141 vs 93 for thr), and others argue that this is not necessarily so,(32) we have examined the conformational space available to a freely rotating chromophore in yfp (pdb code 1myw) with the phenol of tyr66 in both the protonated and unprotonated forms . Both table 1 and figure 8 show that the chromophore (in the phenolic form) in gfp and yfp has differing rotational freedom . In yfp the chromophore has approximately 1.2 times more dihedral freedom than in gfp and although the dihedrals undergo negatively correlated hula - twisting in both proteins, in yfp the average conformation sampled is further from planarity than that found in gfp; i.e., the majority of the sampled conformations are located in quadrant iv . Plot of the vs dihedral angles (see figure 1 for nomenclature) for the 2000 structures obtained in the freely rotating molecular dynamics simulation of gfp (dark blue) and yfp (pink) in their phenolic forms . The majority of the yfp conformations sampled are nonplanar (i.e., in quadrant iv <0 and> 0). During the entire simulation the hydrogen bonds between tyr66 and ser205, arg96 and the imidazolone carbonyl, as well as between tyr203 and water354 remain stable, while those between tyr203 and gln69 were less stable . We also examined the distance between the centroid of tyr66 (the choromophore) and tyr203 . The distance is fairly short and does not change much during the simulation (minimum = 3.43, maximum = 4.18, average = 3.75, sd = 0.103) while the angle between the planes of the phenols varies between 0.0 and 22.5 (average = 6.9, sd = 4.11). The short distance between the phenol rings is indicative of stacking, which may be responsible for the red shift observed in yfp; however, the variability in the angle between the planes of the phenols shows that tyr203 does not significantly restrict the rotational freedom of the excited chromophore . The cambridge structural database (csd)(77) v5.29 comprises 436 384 small molecules crystal structures . It has 5551 structures with two phenol rings in separate molecules that have centroids within 5.00 of each other . The closest ones are 3.16 from each other, and only 832 structures are within 3.75; i.e., the average distance between the phenol centroids in the freely rotating yfp . A plot of the distance vs the angle between the phenol planes in our freely rotating simulation and the csd structures shows that the protein matrix of yfp restricts the distance between tyr66 and tyr203 from being larger than 4.18, but that the angles between the two phenols have as much freedom in yfp as in the small molecule structures with intermolecular distances of less than 4.18 (see figure 9). It does not seem as if the increased fluorescence lifetime of yfp is due to a decrease in the dihedral freedom of the chromophore . It is more likely due to electronic effects such as those proposed by jung et al. (78) they suggested that the anionic form of the chromophore can be described by two mesomeric forms, the benzoidal and the quinoidal structure . The benzoidal form with the majority of its negative charge on the phenol is stabilized by hydrogen bonding to thr203, which is not possible in yfp and therefore the t203y mutation favors the quinoidal form, which may have a longer fluorescence lifetime. (78) distance vs angle between the phenol planes in yfp () and all structures in the csd that have two unconnected phenols separated by less than 5.00 (+). Some have suggested that the crystal structures of strongly fluorescent gfp and gfp - like proteins have their chromophores in a cis configuration, while those of the nonfluorescent proteins are in a trans configuration . However, the trans - planar form of eqfp611 is an important exception: it is fluorescent. (82) remington et al . Have suggested that coplanarity might be more important than isomer configuration in determining the efficiency of fluorescence . Most current theories (see) suggest that a restriction in the rotational freedom of the fluorescent protein chromophore will lead to an increase in fluorescence brightness and/or quantum yield . Our calculations show that this is the case for the systems examined . For bfp, a5, yfp, and gfp, there is an inverse correlation between the dihedral freedom of the chromophore and the quantum yield; see table 1 . In all simulations, the protein matrix is not complementary with a planar chromophore, and in all cases the freely rotating chromophore undergoes a negatively correlated hula twist (also known as a bottom hula twist mechanism).

Fnas represent an advantageous, effective means to obtain diagnostic cellular material as they can often sample a wide area of the target lesion, acquire tumor cells with lower contamination by background stromal connective tissue elements, and allow for immediate assessment during rapid on - site evaluation (rose). During the on - site assessment, a member of the cytopathology team is present and prepares direct smears, using the contents expelled from the needle, at the location of the procedure . This is advantageous for three reasons: each needle pass can be examined to determine tumor cell adequacy; there is an opportunity to engage the clinical care provider in a conversation regarding the preliminary diagnosis and relevant molecular diagnostic tests; and the cytopathology team member can help ensure that the specimen is processed in a manner that optimizes judicious triage for ancillary tests, including molecular studies . Fnas can be performed without rose and in this setting, the contents of the fna needles are typically expelled and rinsed in a cell preservative solution for use in liquid - based cytology (lbc) preparations . Examples of lbc preparations include thinprep (hologic, bedford, ma, usa), surepath (becton dickinson, franklin lakes, nj, usa), thin - layer advanced cytology assay system (tacas, mbl, tokyo, japan), and liqui - prep (lgm international inc ., nonetheless, rose is an effective means to maximize the chances of success in acquiring adequate tumor cells for diagnosis and anticipated molecular studies . The ultimate goal is to prevent unnecessary repeat procedures to obtain additional tissue just for molecular studies which can lead to delays in treatment . For patients afflicted with lung cancer at our institution, we perform rose for cytopathologist - performed fnas and for ebus - guided fnas, performed by our clinical colleagues . The contents of each needle pass are expelled onto a slide which is utilized to prepare direct smears . The needle is then rinsed into buffered media; we utilize rpmi media for this purpose . Commonly, a pair of direct smears is prepared per needle pass; one is air - dried and the other is immediately alcohol - fixed . The air - dried smear is stained on - site with the diff - quik (romanowsky) stain and the stained slide can be examined under the microscope immediately thereafter . The alcohol - fixed smear is stained later in the cytopathology laboratory with the papanicolaou stain . Alternatively, the needle contents can be distributed over multiple smears allowing for flexibility in the utilization of direct smears for cytomorphologic evaluation and ancillary studies (fig . Based on the findings in the diff - quik stained smears, the determination can be made to perform additional needle passes to obtain more tumor cells for diagnosis and/or anticipated ancillary studies while the patient is still accessible . For instance, additional direct smears can be prepared and/or dedicated needle passes for the rinse solution (i.e., the needle contents are rinsed in the rpmi solution without the preparation of smears for those needle passes) obtained for the cell block preparation . This overall approach is flexible, forgiving, and engages the cytopathology team in maximizing the chance of success in obtaining adequate cellular material for diagnosis and ancillary studies . This approach can help minimize the chances of encountering the scenario in which diagnostic cellular material is present on only one smear; microdissection of the cells for molecular studies can result in sacrificing this only diagnostic slide in this context, which can have medico - legal consequences . If this scenario is encountered, however, this risk can be mitigated by digital archiving prior to microdissection either via digital slide scanning and/or obtaining photomicrographs . After immediate assessment of the cytomorphologic findings on the diff - quik stained smears, a preliminary diagnosis can be rendered by the cytopathologist and communicated to the clinical care providers . After the conclusion of the procedure, the needle rinse solution, containing a suspension of aspirated cells, is centrifuged in the cytopathology laboratory to pellet the cells . Once the supernatant is removed, the cell pellet is congealed in a matrix; an agar - like substance such as histogel (thermo scientific, waltham, ma, usa) can be used or the cells can be mixed with plasma and thrombin to create a clot . At our institution, we employ histogel for this purpose . Commonly, this cassette is fixed in formalin and processed to ultimately create a formalin - fixed, paraffin - embedded (ffpe) cell block . This is analogous to a ffpe block of small biopsy tissue, which can be sectioned for evaluation via the hematoxylin and eosin stain and ancillary tests such as immunocytochemistry or molecular diagnostic assays . It should be noted that heavy metal fixatives such as zenker s fixative and acidzinc - formalin, acidic fixatives such as bouin s solution, or decalcification solutions should generally be avoided as these render specimens unusable for molecular testing . The main advantage of using cell blocks is that the majority of ancillary tests are validated for ffpe sections; ffpe cell blocks are treated similarly to traditional surgical pathology ffpe blocks . Furthermore, multiple serial sections from cell blocks can be utilized to perform a battery of ancillary studies . First, adequate cellularity in cell blocks is not guaranteed at the time of the procedure . Performing dedicated needle passes, for the needle rinse solution during the fna procedure, may improve the chances of obtaining a sufficiently cellular cell block but still does not guarantee success . In addition, for hypocellular cell blocks, there is a risk of depleting the cells of interest upon deeper sectioning for molecular tests . Second, the cell block is derived from a needle rinse solution which is a pooled specimen that contains contents of all the needle passes . Thus, if one or more needle passes contain tumor cells but other needle passes contain abundant benign background cellular elements, the tumor cells will be diluted by these benign cells in the cell block . In addition to the above disadvantages, dna extracted from ffpe cell blocks may produce sequencing artifacts as formalin fixation leads to the crosslinking of nucleic acids and proteins and the possibility of sequence alterations . Recently, given the shortcomings of cell blocks, there has been an increasing appreciation of alternative cytopreparatory platforms for molecular testing such as direct smears and lbc samples . In the subsequent sections below, the utilization of these cytopreparatory platforms will be discussed in the context of the clinically relevant mutations and translocations analyzed during molecular diagnostic testing of nsclcs . The epidermal growth factor receptor (egfr) gene encodes a transmembrane growth factor receptor that exhibits tyrosine kinase activity . Upon activation, intracellular signaling is mediated by cytoplasmic effectors in the ras - raf - mek - erk, pi3k - akt, and stat pathways . Egfr mutations are predominantly observed in lung adenocarcinomas; the l858r substitution and small in - frame deletions in exon 19 are the most commonly observed mutations and account for up to 90% of all egfr mutations in this setting . These mutations are more commonly associated with east asian ethnicity, female gender, and non - smoking history . However, these are not absolute rules and clinical characteristics should not be used to exclude lung cancer patients from mutation testing . Lung adenocarcinomas harboring these sensitizing mutations in egfr have been shown to respond to egfr tyrosine kinase inhibitors (tkis)., tki therapy has been demonstrated to result in improved progression - free survival, compared to standard chemotherapy, for patients with lung adenocarcinoma harboring egfr mutations [11 - 16]. Egfr mutation analysis is commonly performed via polymerase chain reaction (pcr) and sequencing - based approaches; advances in the development of testing modalities have afforded a multitude of methodologies . Sanger sequencing is considered the gold standard as this involves direct dna sequence acquisition and can provide information regarding the presence of all potential mutations including common, known mutations and novel mutations . Nonetheless, this test requires a relatively higher enrichment of tumor cell dna content in the sample . The typical analytic sensitivity for sanger sequencing is 15%20% mutant allele, which equates to 30%40% tumor cells assuming that the genetic mutation is a heterozygous event without amplification . This can be problematic in both small biopsy and cytology specimens, especially cell blocks, in which the tumor cell population can be diluted by background benign cellular elements such as inflammatory cells, bronchial epithelial cells, and/or stromal mesenchymal cells . Especially in this setting, a negative mutation result can be either due to the true absence of the mutation in the tumor cells or insufficient percent tumor cellularity that falls below the analytic sensitivity threshold thereby resulting in the failure to detect the mutation even despite the presence of the mutation . Therefore, often times, there is more reliance on tumor cell enrichment by either macrodissection or microdissection to obtain a reliable result . Sanger sequencing is also relatively more labor intensive and time consuming than targeted methods and can lead to longer turnaround times . In contrast to the general sanger sequencing approach, targeted mutation detection methods such as pcr - restriction fragment length polymorphism, real - time pcr, pyrosequencing, high resolution melting analysis (hrma), and pcr fragment analysis can be utilized . The advantages of these approaches include their improved analytic sensitivity and less time - consuming nature leading to reduced turnaround times . At our institution, we utilize a multiplex pcr fragment analysis assay for egfr mutation testing; this allows for the simultaneous assessment of the two most commonly observed egfr mutations (fig . 2). The analytic sensitivity of this method is better than that of sanger sequencing; a minimum of only 10% tumor cells is required . In the past decade, myriad studies have been reported demonstrating that a variety of cytologic samples and cytopreparatory platforms can be effectively utilized for egfr mutational analysis . These have been reviewed elsewhere but salient examples will be discussed below . As mentioned previously, ffpe cell blocks represent the traditional cytopreparatory platform on which ancillary molecular diagnostic tests are performed . Much of the reasoning behind this lies in that ffpe cell blocks best approximate ffpe blocks containing tissue specimens and the majority of the molecular assays are validated using ffpe material . Nonetheless, in light of the inherent disadvantages of cell blocks mentioned previously, investigation into other cytopreparatory platforms for molecular diagnostic assays have been performed by our group and many others . Cytopathology specimen preparation is diverse and versatile; the consequence of this is that each type of cytologic analyte platform needs to be carefully validated for any given molecular test . Cytologic direct smears may ultimately prove to be the cytologic platform best suited for pcr - based analysis due to the high quality of nucleic acids and immediate nature of specimen assessment for tumor cell adequacy . Cells on direct smears are not exposed to formalin but rather undergo an alcohol - based fixation process prior to staining; thus, the negative effects of formalin fixation on nucleic acid quality is non - contributory for direct smears . Furthermore, the tumor cells of interest can be directly visualized on the direct smear and isolated for nucleic acid extraction and molecular analysis; in essence, what you see is what you get . We have previously demonstrated that uncoverslipped and previously coverslipped / decoverslipped diff - quik stained smears are robust sources of cellular material for pcr - based mutational analysis such as egfr mutation testing of lung adenocarcinoma and braf mutation testing of metastatic melanoma fna specimens . We and others have observed that destaining the smears is not necessary for successful dna extraction and pcr . Our preference in utilizing diff - quik stained smears over papanicolaou stained smears is rooted in the following observations: (1) cellular material on a diff - quik stained smear can be easily visualized without a coverslip and immediately triaged for molecular diagnostic testing; and (2) the report by killian et al . That nucleic acids extracted from diff - quik stained smears show better preservation and integrity than dna extracted from papanicolaou stained smears . With respect to the latter point, others have pointed out that papanicolaou stained smears are just as feasible for pcr - based molecular tests . Multiple groups have successfully demonstrated the utilization of cytologic direct smears for egfr mutation testing of lung adenocarcinomas [2,9,25 - 36] with low failure rates and high degree of concordance with egfr molecular testing results from corresponding histologic specimens, when available . In our experience, 100 to 200 tumor cells are sufficient for successful dna isolation and egfr mutation analysis and this is congruent with the observations from others . Lbc samples have also been investigated for egfr mutation testing and have been shown to be an effective analyte for this purpose . This is valuable when a member of the cytopathology team is not available for rose . Several lbc technologies have been developed including thinprep, surepath, tacas, and liqui - prep . Of these, the emerging literature, to date, on the utilization of lbc samples for molecular testing of nsclc have predominantly focused on cytolyt (cytyc corp ., marlborough, ma, usa) cell suspensions and thinprep slides . Collecting fna samples in the methanol - based cytolyt solution results in reduction of background blood as the cytolyt solution exhibits hemolytic properties . The cell suspension can be divided and an aliquot is used to prepare a thinprep slide that is stained via the papanicolaou method, similar to alcohol - fixed papanicolaou stained direct smears . Tumor cells can be isolated from the thinprep slide and used for dna isolation and egfr mutation analysis . Furthermore, the cell suspension aliquot that is not used to prepare a thinprep slide can be centrifuged and the cell pellet itself can be used as an analyte for mutation testing [38 - 40]. Similar to the needle rinse cell suspension utilized to prepare cell blocks, the cytolyt cell suspension is a pooled specimen if multiple needle passes are expelled and rinsed into cytolyt . Thus, if one or more needle passes are high in tumor cell content whereas other needle passes contain a high proportion of benign cellular elements, the tumor cells in the final pooled cell suspension will be diluted . First, targeted methods for detecting egfr mutations with improved analytic sensitivity, relative to sanger sequencing, can be utilized . Second, tumor cell enrichment from the thinprep slide can be accomplished by laser capture microdissection (lcm). To illustrate these points, malapelle et al . Directly compared the performance of egfr mutation analysis by sanger sequencing using paired samples for each case analyzed: (1) pelleted cells from the cytolyt suspension and (2) tumor cells obtained from the thinprep slide via lcm . They observed that egfr mutations were more reliably identified in the latter rather than the former . This difference was minimized when utilizing more sensitive egfr mutation analytic approaches which included hrma and pcr fragment analysis assays . Based on these studies, the authors speculated that coupling the use of highly sensitive egfr mutation detection assays and cytolyt derived cell pellets may be sufficient obviating the need for microscopy . Nonetheless, it must be emphasized that any negative molecular testing result should carefully be reconciled with the analyte input utilized for the mutation assay . This is essential to determine whether the negative result represents a true negative or potentially a false - negative, necessitating possible retesting . In this regard, the utilization of microscopy still remains an essential pre - analytic quality assurance activity to best ensure that the input analyte is of sufficient tumor cellularity to maximize confidence in the results of the mutation assay . Of note, a follow - up study by bellevicine et al . They observed that the direct smears exhibited significantly higher cellularity than thinprep slides, on average . Accordingly, the average yield of dna extracted from direct smears was significantly higher than that from thinprep slides . As the l858r substitution and deletions in exon 19 represent approximately 90% of all egfr mutations in lung adenocarcinoma, one antibody is specific for the exon 21 l858r mutation and the other specific for the 15-base pair/5-amino acid deletion (e746_a750del) in exon 19 (clone 6b6). The immunohistochemical approach to detecting mutant egfr proteins using these antibodies has been examined in several studies on lung cancer tissues as well as cytologic and small biopsy samples [42 - 48]. The sensitivity of these assays range from 47%92% but their high positive predictive value and specificity supports the feasibility of utilizing this approach as a first - line screening approach . First, immunohistochemistry should be performed after careful validation and formulation of immunostain scoring criteria . The significance of how best to interpret equivocal staining results should be clarified as overinterpreting weak immunoreactivity as a positive mutation result can lead to increased false positives and decreased specificity . Second, the clone 6b6 antibody best detects the 15-base pair (e746_a750del) egfr deletion mutant protein but demonstrates variable immunoreactivity for the egfr mutant proteins resulting from non-15-base pair deletions in exon 19 . The anaplastic lymphoma kinase (alk) protein is a receptor tyrosine kinase and rearrangements involving the alk gene locus is observed in approximately 5% of lung adenocarcinomas . These mutations are more commonly observed in younger age patients who are never - smokers . However, there can be exceptions to this and clinical characteristics should not be used to exclude lung cancer patients for alk rearrangement testing . Most alk rearrangements in lung adenocarcinoma result from interstitial deletions and small inversions within the short arm of chromosome 2 . This results in the fusion of portions of the echinoderm microtubule - associated protein - like 4 (eml4) and alk genes . Other less common alk rearrangements involve fusions between alk and other genes, such as kif5b and tfg . Alk rearranged lung adenocarcinoma has been recognized as a legitimate target for small molecular inhibitor therapy; crizotinib was shown to be efficacious in treating patients with these cancers . In a phase 1 study evaluating 143 patients, an overall response rate of 61% and estimated overall survival rates of 74.8% at 12 months therefore, the evaluation of lung adenocarcinoma fna samples for alk rearrangements, in addition to egfr mutations, has become increasingly incorporated into patient management algorithms . Fish is currently the preferred approach to assaying lung adenocarcinomas for alk rearrangements according to expert recommendations based on review of the literature . In the united states of america, there is only one test approved by the food and drug administration (fda) for this purpose . This assay utilizes a dual alk breakapart probe strategy in which orange and green labeled probes hybridize to the highly conserved translocation breakpoint region in the alk gene . Alk gene loci that have not undergone rearrangement typically display fused orange and green signals (yellow) or juxtaposed touching orange and green signals . When an alk rearrangement occurs, the orange and green signals become separated . However, as the majority of the alk rearrangements involve a small inversion within chromosome 2p, rather than a rearrangement involving another chromosome, the extent to which the two signals are split is finite . In order to score a nucleus as positive for the alk rearrangement, the orange and green signals must be separated by a distance of> 2 signal diameters (fig . 3); a nucleus can also be scored positive if a single orange signal without a corresponding green signal is observed . Typically, up to 100 tumor cell nuclei are scored in this assay and a lung adenocarcinoma is considered positive for the alk rearrangement if at least 15% of the nuclei are scored as positive for the rearrangement . The fda has approved this dual breakapart probe set for use on paraffin embedded tissue sections . As mentioned previously, paraffin embedded cell blocks are processed similarly to paraffin embedded tissue blocks; thus, cell blocks are traditionally used for the performance of alk rearrangement fish assays . Nonetheless, there is an observed noticeable failure rate when using and relying on cell blocks for these assays due to insufficient tumor cell material in a significant proportion of cases . Thus, we and others have investigated alternative cytopreparatory platforms for alk fish . In our study, we demonstrated the effective use of diff - quik stained direct smears for this purpose and observed that the performance of this assay using direct smears was better than the performance using cell blocks . . Also examined the use of papanicolaou stained direct smears and observed that the failure rate due to insufficient cellularity was significantly higher for cell blocks than for direct smears . Therefore, the utilization of both diff - quik and papanicolaou stained smears are feasible analytes for alk fish . Recently, thinprep slides have also been shown to be a feasible platform for alk fish . The advantage of utilizing cytologic preparation platforms such as direct smears and thinprep slides for this purpose is that entire tumor cell nuclei are being analyzed . Fish evaluation on paraffin sections derived cell blocks and other ffpe blocks are prone to signal loss in some of the tumor cells due to section truncation artifacts . In addition, alternative methods to fish have been the subject of recent investigation . Immunohistochemistry utilizing antibodies directed against alk is an attractive alternative as it is simpler, quicker, and less expensive . The challenge associated with this approach is that the alk protein is expressed at much lower levels in alk rearranged lung tumors than in anaplastic large cell lymphoma, the prototypical alk rearranged tumor . Fortunately, monoclonal anti - alk antibodies (clones d5f3, d9e4, and 5a4) have been shown to exhibit high sensitivity and specificity . Immunohistochemistry using the d5f3 and 5a4 monoclonal antibodies on cytologic specimens have been recently described and shown to exhibit a high degree of concordance with alk fish testing . This supports the feasibility of utilizing an immunohistochemical approach as a first - line screening methodology to select specimens for alk fish testing . Of note, the mouse monoclonal anti - alk1 antibody (cd246) typically used for the diagnosis of anaplastic large cell lymphoma is less reliable for identifying alk rearrangements in lung adenocarcinoma; this is most likely attributable to the limited sensitivity of this particular antibody in detecting the lower expression levels of the alk fusion proteins in lung adenocarcinoma relative to anaplastic large cell lymphoma . Finally, the application of reverse transcriptase pcr (rt - pcr) to cytologic direct smears for alk rearrangement analysis has been recently reported . When evaluating a cohort of paired cytologic - histologic specimens by rt - pcr, mitiushkina and nonetheless, as a cautionary note, there is a high degree of variability in eml4-alk fusion events with at least 13 variants of eml4-alk being reported . Furthermore, other fusion partners to alk such as tfg and kif5b can be observed . Therefore, an rt - pcr approach may not capture all clinically relevant alk rearrangements . While egfr mutations and alk rearrangements represent the two best characterized, clinically actionable molecular alterations in nsclc, other molecular markers are becoming increasingly appreciated and investigated . Ros1 encodes a receptor tyrosine kinase and is rearranged in approximately 2% of lung adenocarcinomas . Met encodes another receptor tyrosine kinase, hepatocyte growth factor receptor, and is amplified in a subset of nsclc; a significant proportion of these cases are seen in context of acquired resistance to egfr tkis . As crizotinib also targets this receptor, studies are under way to determine whether this agent will be effective in met amplified lung cancers . Therapeutic agents targeting braf, such as dabrafenib, are currently being investigated in clinical trials . In addition, gene rearrangements involving the ret tyrosine kinase gene (e.g., kif5b - ret) have been observed in approximately 2% of lung adenocarcinomas; ret specific tkis such as sunitinib, sorafenib, and vandetanib may be useful in treating patients with these lung cancers . Finally, amplification of fgfr1 and mutations in pik3ca have been observed in some lung squamous cell carcinomas and agents targeting these gene products are also under investigation . The above mentioned molecular genetic markers highlight the distinct molecular profiles that are becoming increasingly appreciated for different subtypes of nsclc, especially adenocarcinomas and squamous cell carcinomas . Therefore, efforts to correctly subtype cases of nsclc are important, including challenging cases of poorly - differentiated nsclc . At our institution, if the subtyping of nsclc is deemed to be challenging based on cytomorphologic evaluation alone, immunocytochemistry is utilized; we have demonstrated that this can be accomplished using smears as well as cell blocks . For example, napsin - a and thyroid transcription factor-1 are often positive in lung adenocarcinomas and can be utilized for confirming this subtype of nsclc . In contrast, p63 and p40 are useful markers for confirming a diagnosis of squamous cell carcinoma . Ultimately, utilizing fna samples of nsclc for immunocytochemistry must be judiciously leveraged to ensure that sufficient material still exists for molecular ancillary testing . Given the continuously evolving landscape in our understanding of the genetic events responsible for lung cancer pathogenesis, the integration between anatomic pathology, particularly cytopathology, and molecular diagnostics will become even more essential . Emerging molecular technological advances, such as next generation sequencing (ngs), that allow for high - throughput, high - sensitivity molecular analyses will likely play an important role in the management of patients with nsclc . Cytologic specimens, based on recent reports, represent a robust source of cellular material for ngs [62 - 65].

These tumors are classified as typical net (tnet), goblet cell carcinoid (gcc), and atypical gcc histologies . Ex - goblet or composite - goblet are further classified into signet ring cell carcinoid (srcc) and poorly differentiated adenocarcinoid of the appendix . These tumors have a distinctive morphology showing tight clusters of cells with compact nuclei and abundant intracytoplasmic mucin resembling goblet or signet ring cells often with admixed enterochromaffin cells . Pathologic features of gcc include presence of large mucin filled cells with crescent nuclei arranged in small clumps or rosettes mixed with cells of typical carcinoid appearance that stain positive for chromogranin a . Patients with tnet have a 5-year survival ranging from 60% to 84%, with the most common site of metastasis being the liver . On the other hand, atypical gcc have a more aggressive clinical course with an increased incidence of lymph node and distant metastases, along with a lower 5-year survival ranging from 36% to 56% [5 - 7]. Current management of gcc and atypical gcc is based on very limited data from small single institutional experiences . The rarity of appendiceal net, gcc, and srcc limits the ability to conduct appropriate randomized clinical trials to explore the optimal management . To assess the role of clinicopathologic features in survival of net, gcc, and srcc patients, the outcome of cases reported to the national cancer institute s surveillance epidemiology and end results (seer) program was evaluated . Furthermore, to characterize the management of net, gcc, and srcc, a treatment strategy based on results of current analysis, published literature and institutional experience is suggested . Seer data collected between 1973 and 2011 was used to identify cases with appendiceal tnet, gcc, and srcc . The seer registry data collection began in the early 1970s and gradually expanded from the original nine to the current 18 registries that now account for over a quarter of the united states population . Eligibility criteria included international classification of diseases for oncology third edition (icd - o-3) codes for primary site of appendix and histologic types of tnet (8240, 8241, 8242, 8246, and 8270), gcc (8243, 8244, 8245, and 8249), and srcc (8490). Information regarding age at diagnosis, sex, race, year of diagnosis, grade, histology, tumor location, type of surgery, vital status and duration of follow - up, was extracted from the seer database . The patients characteristics were compared among three histologic types: tnet, gcc, and srcc by chi - square test for race, sex, and stage, and kruskal - wallis test for age . Survival functions were estimated by the kaplan - meier method and log - rank test was used to assess the difference in overall survival (os) between three histologic types: tnet, gcc, and srcc . Univariate survival analysis for each covariate was carried out using the cox proportional hazards model . The multivariate survival analysis of histology was conducted after adjusting for age, race, and sex using a backward variable selection method with an alpha level of removal of 0.1 . The model was stratified by stage since there was an interaction effect between histology and stage on os . 9.3 (sas institute inc ., cary, nc) was used for data analyses . Null hypotheses of no difference were rejected if p - values were less than 0.05, or, equivalently, if the 95% confidence intervals (cis) of risk point estimates excluded 1 . Seer data collected between 1973 and 2011 was used to identify cases with appendiceal tnet, gcc, and srcc . The seer registry data collection began in the early 1970s and gradually expanded from the original nine to the current 18 registries that now account for over a quarter of the united states population . Eligibility criteria included international classification of diseases for oncology third edition (icd - o-3) codes for primary site of appendix and histologic types of tnet (8240, 8241, 8242, 8246, and 8270), gcc (8243, 8244, 8245, and 8249), and srcc (8490). Information regarding age at diagnosis, sex, race, year of diagnosis, grade, histology, tumor location, type of surgery, vital status and duration of follow - up, was extracted from the seer database . The patients characteristics were compared among three histologic types: tnet, gcc, and srcc by chi - square test for race, sex, and stage, and kruskal - wallis test for age . Survival functions were estimated by the kaplan - meier method and log - rank test was used to assess the difference in overall survival (os) between three histologic types: tnet, gcc, and srcc . Univariate survival analysis for each covariate was carried out using the cox proportional hazards model . The multivariate survival analysis of histology was conducted after adjusting for age, race, and sex using a backward variable selection method with an alpha level of removal of 0.1 . The model was stratified by stage since there was an interaction effect between histology and stage on os . 9.3 (sas institute inc ., cary, nc) was used for data analyses . Null hypotheses of no difference were rejected if p - values were less than 0.05, or, equivalently, if the 95% confidence intervals (cis) of risk point estimates excluded 1 . The seer database yielded 1,021 tnet patients, 1,582 with gcc, and 534 srcc patients . Between 1973 and 2011, the incidence of tnet, gcc, and srcc increased . In 2011, the incidence of tnet reached 6.7, gcc was 0.3, and srcc was two patients per 100,000 persons . Baseline characteristics are compared between appendiceal tnet, gcc, and srcc in table 1 . Significant differences in age at presentation (p <0.001), sex distribution (p <0.001), surgery (p <0.001), type of surgery (appendectomy, right hemicolectomy, and surgery not otherwise specified) (p <0.001), and stage (p <tnet patients presented younger (median age of 41 vs. 54.5 in gcc and 57 years in srcc). Female patients constituted a higher proportion among tnet cases (66.2% vs. 61.4% of srcc and 48.5% of gcc). Advanced stage disease was more common in srcc patients (60.9% vs. 14.1% in tnet and 10.4% in gcc). A higher proportion of white patients was observed in all three histologies, but the difference was not statistically significant (p=0.11). Surgery was performed in 839 (82.6%) of the tnet patients, 1,365 (86.4%) of the gcc, and 419 (78.6%) of the srcc patients, and these differences were statistically significant (p <0.001). Appendectomy was performed in 52 of patients with tnet (5.9%) as compared to 54 of the gcc (3.9%), and 9 of the srcc (2.1%). Right hemicolectomy was performed in 484 of the tnet (54.9%), 1,208 of the gcc (86.4%), and 301 of the srcc patients (70.7%). The differences in type of surgery were significantly different among the three histologies (p <0.001). The mos for gcc and tnet were not reached at the time of this analysis . Comparing survival among the different histologies, tnet had a survival advantage over gcc (hazard ratio [hr], 0.56; 95% ci, 0.45 to 0.69; p=0.005), which had a better survival over srcc histology (hr, 0.22; 95% ci, 0.19 to 0.26; p <0.001) (table 3). In each stage, tnet and gcc had significant survival advantages over srcc (figs . 1 - 3). In the localized stage, mos was not reached for any of the three histologies . Comparing srcc, the hr for survival was 0.26 (0.15 - 0.46; p <0.001) for tnet and 0.42 (0.26 - 0.69; p <0.001) for gcc . For regional stage disease, the mos for srcc was 35 months (95% ci, 30 to 45), while mos for gcc and tnet were not reached . The tnet regional disease stage patients had survival advantage over gcc (hr, 0.37; 95% ci, 0.24 to 0.59; p <0.001). Gcc had better survival compared to srcc patients with regional disease stage (hr, 0.29; 95% ci, 0.21 to 0.40; p <0.001). For distant disease stage, mos was 32 (95% ci, 13 to not reached) for tnet, 23 (95% ci, 18 to 28) for gcc, and 15 months (95% ci, 13 to 18) for srcc . In this group, tnet patients had survival advantage over gcc (hr, 0.61; 95% ci, 0.44 to 0.83; p=0.002). Gcc patients survived better compared to srcc patients with distant disease (hr, 0.73; 95% ci, 0.60 to 0.90; p=0.003). After adjusting for age, stage, and histology, there was a statistically significant difference in survival for srcc patients treated with hemicolectomy compared to appendectomy (p=0.01). There was no significant difference in survival for tnet and gcc patients based the type of surgery (p=0.21 and p=0.94, respectively). In the multivariate analysis stratified by stage and age, there was a statistically significant difference in survival favoring tnet (hr, 0.41; 95% ci, 0.31 to 0.55) and gcc (hr, 0.59; 95% ci, 0.48 to 0.72) over srcc . The seer database yielded 1,021 tnet patients, 1,582 with gcc, and 534 srcc patients . Between 1973 and 2011, the incidence of tnet, gcc, and srcc increased . In 2011, the incidence of tnet reached 6.7, gcc was 0.3, and srcc was two patients per 100,000 persons . Baseline characteristics are compared between appendiceal tnet, gcc, and srcc in table 1 . Significant differences in age at presentation (p <0.001), sex distribution (p <0.001), surgery (p <0.001), type of surgery (appendectomy, right hemicolectomy, and surgery not otherwise specified) (p <0.001), and stage (p <tnet patients presented younger (median age of 41 vs. 54.5 in gcc and 57 years in srcc). Female patients constituted a higher proportion among tnet cases (66.2% vs. 61.4% of srcc and 48.5% of gcc). Advanced stage disease was more common in srcc patients (60.9% vs. 14.1% in tnet and 10.4% in gcc). A higher proportion of white patients was observed in all three histologies, but the difference was not statistically significant (p=0.11). Surgery was performed in 839 (82.6%) of the tnet patients, 1,365 (86.4%) of the gcc, and 419 (78.6%) of the srcc patients, and these differences were statistically significant (p <0.001). Appendectomy was performed in 52 of patients with tnet (5.9%) as compared to 54 of the gcc (3.9%), and 9 of the srcc (2.1%). Right hemicolectomy was performed in 484 of the tnet (54.9%), 1,208 of the gcc (86.4%), and 301 of the srcc patients (70.7%). The differences in type of surgery were significantly different among the three histologies (p <0.001). The mos for gcc and tnet were not reached at the time of this analysis . Comparing survival among the different histologies, tnet had a survival advantage over gcc (hazard ratio [hr], 0.56; 95% ci, 0.45 to 0.69; p=0.005), which had a better survival over srcc histology (hr, 0.22; 95% ci, 0.19 to 0.26; p <0.001) (table 3). In each stage, tnet and gcc had significant survival advantages over srcc (figs . 1 - 3). In the localized stage, mos was not reached for any of the three histologies . Comparing srcc, the hr for survival was 0.26 (0.15 - 0.46; p <0.001) for tnet and 0.42 (0.26 - 0.69; p <0.001) for gcc . For regional stage disease, the mos for srcc was 35 months (95% ci, 30 to 45), while mos for gcc and tnet were not reached . The tnet regional disease stage patients had survival advantage over gcc (hr, 0.37; 95% ci, 0.24 to 0.59; p <0.001). Gcc had better survival compared to srcc patients with regional disease stage (hr, 0.29; 95% ci, 0.21 to 0.40; p <0.001). For distant disease stage, mos was 32 (95% ci, 13 to not reached) for tnet, 23 (95% ci, 18 to 28) for gcc, and 15 months (95% ci, 13 to 18) for srcc . In this group, tnet patients had survival advantage over gcc (hr, 0.61; 95% ci, 0.44 to 0.83; p=0.002). Gcc patients survived better compared to srcc patients with distant disease (hr, 0.73; 95% ci, 0.60 to 0.90; p=0.003). After adjusting for age, stage, and histology, there was a statistically significant difference in survival for srcc patients treated with hemicolectomy compared to appendectomy (p=0.01). There was no significant difference in survival for tnet and gcc patients based the type of surgery (p=0.21 and p=0.94, respectively). In the multivariate analysis stratified by stage and age, there was a statistically significant difference in survival favoring tnet (hr, 0.41; 95% ci, 0.31 to 0.55) and gcc (hr, 0.59; 95% ci, 0.48 to 0.72) over srcc . The analysis of the seer registry sample confirms the differences in presentation and outcome of these diseases . Tnet tend to occur at a younger age and has a less aggressive clinical course, early stage at presentation and significantly improved os . At the other end of the disease spectrum, the aggressive nature of srcc is reflected by the high risk of distant metastasis at diagnosis, and worse survival outcomes even when controlled for stage . The reported sex distribution in tnet ranges from a female preponderance (63% to 73%) to evenly distributed . Similarly for gcc, literature reports range from female gender predominance, to equal distribution . Most of these series are based on small numbers of patients from single institution studies . The current report confirms, using a large national database, that there are gender differences in distribution of tnet, gcc, and srcc (p <0.001). For srcc histology, we confirmed a higher prevalence amongst women in this seer analysis as compared to gcc histology, consistent with a previous report . Additionally, appendiceal srcc appear to be clinically similar to right - sided, microsatellite unstable, signet ring cell colon adenocarcinoma with respect to gender distribution, age of diagnosis and outcome . This raises the question whether appendiceal srcc and right colon adenocarcinomas behave more like a single disease entity distinct from other colorectal cancer (crc) adenocarcinoma and appendiceal gcc . This apparent difference in clinical behavior will require further characterization through genomic analysis of crc srcc, appendiceal srcc and typical gcc . Hence, a molecularly based classification may facilitate prognostication and perhaps target identification for future clinical trials . The analysis of the seer data confirms that histology and stage at presentation are the major determinants of outcome . The high risk of metastatic disease in the srcc patients confirms the aggressive clinical behavior of this tumor . The rate of metastasis at diagnosis in srcc histology ranges from 14% to 63% . The survival advantage for tnet and gcc over srcc persisted after controlling for the difference in stage distribution . Similar observations were reported with 5-year survival of patients with advanced stage gcc, srcc and poorly differentiated adenocarcinoid tumor being 100%, 38%, and 0%, respectively . Given their rarity, there is no consensus on the management of appendiceal tnet, gcc, or srcc . The treatments employed included surgical resection and cytoreduction of primary tumor and metastatic sites, intraperitoneal chemotherapy and systemic fluorouracil - based chemotherapy . Debulking surgery and hyperthermic intraperitoneal chemotherapy treatment is not well - established in appendiceal net, gcc, and srcc tumors, although it improves appendiceal mucinous adecarcinoma . Clinically, the management decisions are usually based on histologic subtype and stage, as well as the patient s performance status . Early stage tnet, gcc, or srcc should be managed surgically, although the extent of surgery is an open question . Historically, the accepted surgical dogma was that simple appendectomy is sufficient for resection of tnet less than 2 cm . Recent reports demonstrated that appendiceal tnet measuring less than 2 cm may have regional nodal involvement, raising the question whether a more extensive surgery than simple appendectomy is required irrespective of size . These observations are further supported by a seer analysis where 32.8% of patients with net less than 2 cm in size (20/61) had lymph node metastasis . The north american neuroendocrine tumor society (nanets) and european neuroendocrine tumor society (enets) guidelines suggest right hemicolectomy for all tumors> 2 cm or presence of deep mesoappendiceal invasion, positive or unclear margins, higher proliferative rate (grade 2), angi olymphatic invasion, and mixed histology, irrespective of tumor size . While simple appendectomy may be adequate for early stage gcc, cecal involvement or high mitotic count index should be an indication for right hemicolectomy . Surgery for srcc should always involve a right hemicolectomy due to the high likelihood of lymph node metastasis, irrespective of the size of the primary appendiceal mass . This is supported by our analysis of the seer data that indicates superior survival for patients with srcc who undergo right hemicolectomy . The role of surgical resection of the ovaries in female patients with localized srcc or gcc remains controversial . A summary of the nanets and enets guidelines is provided in table 4 . Unlike crc adenocarcinoma, the role of adjuvant therapy is not established for tnet, gcc, or srcc . Based on our analysis, patients with tnet have an excellent os and therefore adjuvant therapy is not indicated . On the other hand, given the high risk of systemic metastasis in srcc, we recommend offering adjuvant therapy for all patients undergoing resection . In our analysis of the seer sample, the outcome of patients with early stage gcc appears favorable, suggesting that patients with gcc should be considered for adjuvant therapy only if they have high risk features such as cecal invasion, perforation, or lymph node involvement . For gcc patients with advanced stage disease, options of treatments include peritoneal debulking with intraperitoneal chemotherapy for patient with limited peritoneal disease or systemic fluorouracilbased chemotherapy . For srcc patients, our recommendations include treating with systemic fluorouracil - based chemotherapy initially and only consider peritoneal resection for patients with good response to initial therapy . A summary of the proposed treatment algorithm is provided in fig . Predictive and prognostic biomarkers are not well defined for these diseases . In an attempt to identify gene variations between appendiceal mucinous adenocarcinoma and gcc, we profiled nine gcc samples using second - generation gene sequencing . Only one patient with gcc (11%) had kras mutation, and two had tp53 mutation; myc, smad4, and apc mutations were absent in gcc . Proved the absence of kras mutations in 16 and 14 gcc samples . In the light of its rarity, molecular differentiation markers for appendiceal tnet, gcc, and further confirmation with larger studies is needed to evaluate pathologic genomic sequencing for the three histologies in order to detect differences in survival and possible future biomarkers that predict response to specific target treatments . The seer registry findings confirm the distinct clinical entities of tnet, gcc, and srcc with respect to presentation and outcome.

Igg4-rd is a newly recognized fibroinflammatory condition of unknown etiology characterized by tumefactive lesions, dense lymphoplasmacytic infiltration rich in igg4-positive plasma cells, storiform fibrosis and an elevated serum igg4 level [13]. The kidney is one of the organs commonly affected by igg4-rd, and tubulointerstitial nephritis (tin) with infiltration of numerous igg4-positive plasma cells is the predominant type of kidney lesion [47]. High levels of igg and igg4 are striking and characteristic features of renal involvement such as multiple lesions with low attenuation demonstrated by contrast - enhanced computed tomography are often evident [47]. Although tin is the main feature of the kidney lesion in igg4-rd, some glomerular lesions have also been described, and membranous nephropathy (mn) has been recognized as the most common form [48]. In previously reported cases of mn associated with igg4-rd, mn was diagnosed together with, or after the onset of igg4-rd [912]. Here, we report a case of idiopathic mn as a preceding disease before the onset of igg4-rd . He had previously been well and had no significant medical history . His serum total protein level was 46 g / l (4.6 g / dl), serum albumin 17 g / l (1.7 g / dl), urinary protein excretion 4.3 g / day without occult blood and the 24-h creatinine clearance was normal at 1.51 ml / s (90.5 ml / min) (table 1). The anti - nuclear antibody was negative, and serum immunoglobulins g, a and m were all within normal limits at 11.98 g / l (1198 mg / dl), 11.37 complement levels were also within normal limits: c3 1.12 g / l (112 mg / dl), c4 0.314 g / l (31.4 mg / dl) and ch50 30 u / ml . Contrast - enhanced computed tomography (ct) demonstrated small, non - specific nodules in the right lung, and a hemangioma of the liver . Table 1.clinical course and laboratory findings in the present casemay 2008january 2009july 2009july 2010december 2010march 2011august 2011eventdiagnosed as mnworsening of nsdiscontinuation of psladmission to our hospitaltreatmentdiureticspsl, furosemidefurosemide, olmesartanfurosemide, olmesartanfurosemide, olmesartanfurosemide, olmesartanfurosemide, olmesartantp (g / l)46566071778592alb (g / l)17152931303124igg (g / l)12nanananana39urinalysis (protein)3 + 3 + 3 + 3 + 3 + 3 + 3+up (g / day)4.33.03.1nanana2.124 h ccr (ml / s)1.511.121.37nanana1mn, membranous nephropathy; ns, nephritic syndrome; psl, prednisolone; tp, total protein; alb, albumin; up, urinary protein excretion; ccr, creatinine clearance; na, data not available . Clinical course and laboratory findings in the present case mn, membranous nephropathy; ns, nephritic syndrome; psl, prednisolone; tp, total protein; alb, albumin; up, urinary protein excretion; ccr, creatinine clearance; na, data not available . Percutaneous kidney biopsy was performed, and the histology revealed diffuse membranous changes on the glomerular basement membrane without tubulointerstitial lesions (figure 1a and b). An immunofluorescence study showed diffuse, fine - granular staining of igg, c3, fibrinogen, kappa- and lambda - chain in the glomerular capillary loops . There were no deposits in other areas including mesangeal matrix, subendothelial aspects and tubular basement membrane . An additional immunofluorescence study of igg subclasses showed predominant positive staining for igg4 and igg1 and weak positive staining for igg2 and igg3 in the glomeruli (figure 1c). An electron microscopy examination revealed diffuse, homogenous, small electron - dense deposits in the subepithelial aspects of the glomerular basement membrane, which corresponded to ehrenreich and churg stage 2 . Subendothelial deposits were not present, and endothelial cells did not contain any tubuloreticular structure . The mesangial matrix and tubular basement membrane did not contain electron - dense deposits . 1.histopathologic findings of kidney biopsy at the previous hospital at the time of onset of idiopathic mn . (a) light microscopy shows no significant mesangial cell or matrix proliferation in glomeruli, and no interstitial lesions . (b) diffuse, small deposits are evident in the subepithelial aspects of the glomerular basement membrane . (c) an immunofluorescence study of igg subclasses shows predominant positive staining for igg1 and igg4 and weak positive staining for igg2 and igg3 in glomeruli . Histopathologic findings of kidney biopsy at the previous hospital at the time of onset of idiopathic mn . (a) light microscopy shows no significant mesangial cell or matrix proliferation in glomeruli, and no interstitial lesions . Periodic acid - schiff stain, 200 . (b) diffuse, small deposits are evident in the subepithelial aspects of the glomerular basement membrane . (c) an immunofluorescence study of igg subclasses shows predominant positive staining for igg1 and igg4 and weak positive staining for igg2 and igg3 in glomeruli . A systemic work - up revealed no other disease or malignancy, and the patient prednisolone was administered at 40 mg daily, but the patient suffered repeated cerebral infarction attacks after starting the therapy . Prednisolone was tapered and discontinued 6 months after the start of the therapy, without any significant effect . The patient's proteinuria persisted at around 3 g / day and he was followed up while receiving diuretics and the angiotensin receptor blocker, olmesartan . Two years after discontinuation of glucocorticoid therapy, the patient developed epigastric discomfort and was hospitalized again . Laboratory examinations revealed elevated serum levels of ast, alt, alp, total bilirubin and -gtp [64 iu / l, 106 iu / l, 1948 iu / l, 111 mol / l (6.5 mg / dl) and 1544 iu / l, respectively] accompanied by a slight increase in serum creatinine at 100 mol / l (1.13 mg / dl) and a normal serum amylase level of 111 iu / l . Abdominal ct revealed swelling of the pancreatic head, dilatation of the main pancreatic duct and stenosis of the common bile duct . Tumor markers including cea and ca19 - 9 were all within the normal ranges, while hypergammaglobulinemia was observed and the serum igg level was elevated at 39.21 g / dl (3921 mg / dl) with marked elevation of igg4 at 19.2 hypergammaglobulinemia persisted, and serum iga, igm and complement studies showed that all were within normal limits . Serum anti - nuclear antibody was negative, whereas the rheumatoid factor was positive at 120.6 iu / ml . Urinary protein excretion was 2.1 g / day without occult blood and 24-h creatinine clearance was mildly deteriorated at 1 ml / s (60.0 ml / min) (table 1). Excretion of urinary 2-microglobulin (2-mg) and n - acetyl--d - glucosaminidase was markedly elevated at 11 636 g / day and 25.0 iu / day, respectively . Contrast - enhanced ct showed bronchial thickening in the bilateral lungs, pancreatic swelling with stenosis of the main pancreatic duct and bile duct, bilateral swollen kidneys with multiple lesions exhibiting low attenuation, periaortic and periarterial mass lesions, diffuse swelling of the prostate gland and systemic lymphadenopathy, compatible with the radiologic abnormalities characteristic of igg4-rd (figure 2). 2.contrast-enhanced ct at our hospital at the time of onset of igg4-rd . Marked swelling of pancreatic head and bilateral swollen kidneys with multiple lesions exhibiting low attenuation were observed . Contrast - enhanced ct at our hospital at the time of onset of igg4-rd . Marked swelling of pancreatic head and bilateral swollen kidneys with multiple lesions exhibiting low attenuation were observed . Percutaneous kidney biopsy was not performed since we could not obtain the patient's agreement . Instead, percutaneous needle biopsies from the pancreas and prostate gland specimens obtained by biopsy from the prostate gland and pancreas revealed dense lymphoplasmacytic infiltration with storiform fibrosis and infiltration of numerous igg4-positive plasma cells (igg4/ igg - positive plasma cell ratio> 50%) without malignancy (figure 3). 3.histopathologic features of a specimen obtained from the prostate gland by percutaneous needle biopsy at our hospital at the time of onset of igg4-rd . There is no evidence of cancer cells and there is a typical swirling pattern of fibrosclerosing inflammation with infiltration of numerous lymphocytes, the so - called storiform fibrosis. (b) immunohistochemical staining for igg in a needle biopsy specimen from the prostate gland, 150 . Immunohistochemical staining for igg4 in a needle biopsy specimen from the prostate gland, 150 . The igg4-positive plasma cell / igg - positive plasma cell ratio was over 50% in tissue from the prostate gland . Histopathologic features of a specimen obtained from the prostate gland by percutaneous needle biopsy at our hospital at the time of onset of igg4-rd . There is no evidence of cancer cells and there is a typical swirling pattern of fibrosclerosing inflammation with infiltration of numerous lymphocytes, the so - called storiform fibrosis. (b) immunohistochemical staining for igg in a needle biopsy specimen from the prostate gland, 150 . Immunohistochemical staining for igg4 in a needle biopsy specimen from the prostate gland, 150 . The igg4-positive plasma cell / igg - positive plasma cell ratio was over 50% in tissue from the prostate gland . Western blot analysis of the patient's serum for the detection of serum autoantibodies against the m - type phospholipase a2 receptor (anti - pla2r) was performed, as described previously, and was negative . Oral administration of prednisolone was initiated at 40 mg daily, and the patient's clinical symptoms including jaundice and anorexia improved rapidly . The laboratory data and multiple organ lesions also improved, while proteinuria persisted at around 3 g / day . It has been reported that mn is present in 7% of kidney biopsy specimens in igg4-rd . Most cases of mn associated with igg4-rd are accompanied by igg4-related tin [46, 9, 10], although recent reports have also documented mn without tin but with other features of igg4-rd (8, 11, 12). In mn associated with igg4-rd, positive deposition of igg4 in the glomerular basement membrane has been observed, either predominantly or together with other igg subclasses [812]. Originally, the immunofluorescent examination of igg subclasses was known as a very useful tool to distinguish the primary mn from the secondary mn . Predominant deposition of igg4, often accompanied by other subclasses such as igg1, was one of the well - known features of idiopathic mn . Moreover, idiopathic mn and mn associated with igg4-rd share an essential immunologic feature known as th2 cytokine - mediated immune reaction [14, 15], suggesting that these two disorders share common pathophysiologic processes for increased production of th2 cytokines and subsequent increase of igg4 . On the other hand, these two disorders differ in many other features, including clinical symptoms, laboratory parameters and radiographic findings . An anti - pla2r antibody, which has been described as a major target antigen involved in idiopathic mn and seen in 7080% of patients with idiopathic mn [13, 1618], has been reported to be negative in all cases of igg4-rd and also cases of mn associated with igg4-rd [8, 10, 12], suggesting no involvement of this antibody in the development of igg4-rd . In the present case, there was no evidence of any association of igg4-rd based on the clinical symptoms, serological data and imaging findings when the patient developed nephrotic syndrome . Therefore, it was reasonable to have diagnosed the present case as idiopathic mn at the time of onset of nephrotic syndrome . The anti - pla2r antibody is described as negative in all cases evaluated in mn associated with igg4-rkd and was also negative in this patient, although the result was obtained from the serum 3 years after the diagnosis of idiopathic mn and after corticosteroid therapy . However, 2025% of patients with idiopathic mn are reported to be negative for the antibody [1618]. The absence of the anti - pla2r antibody is thus only partly helpful to distinguish from idiopathic mn . Interestingly, the patient's total serum protein level increased gradually without any significant improvement of proteinuria or elevation of the serum albumin level (table 1), and eventually the clinical symptoms and laboratory abnormalities of igg4-rd became evident 3 years after the onset of idiopathic mn . Accordingly, it is possible that some conditions underlying the persistent predominance of th2-type immune responses during the 3 years of follow - up might have led to the development of igg4-rd . Currently, idiopathic mn is defined as mn without any other disorder that could cause secondary mn, with predominant deposition of igg4 in the glomerular basement membrane . However, mn associated with igg4-rd has also been revealed to be igg4-predominant and, interestingly, the igg subclass of the anti - pla2r antibody seen in idiopathic mn is predominantly igg4 . Although the pathogenesis of igg4-rd is poorly understood, it may share some common mechanisms with idiopathic mn, and thus a comparative study of the two diseases might be helpful for clarifying their pathogenesis . Consideration for igg4-rd in mn, including systemic evaluation or measurement of serum igg4 level, would be necessary in addition to the evaluation of the anti - pla2r antibody and igg subclasses in the immune complex deposited on the glomerular basement membrane.

Treatment duration lasts for months, and is often fraught with potentially dangerous side effects . Some of these side effects, such as influenza - like illness, myalgias, depression, and cytopenias can be intolerable resulting in premature treatment cessation, but have no lasting effects . Ocular complications are among the complications of interferon therapy and include retinal hemorrhages, cotton wool spots, loss of color vision, cataracts, glaucoma, and occasionally retinal artery or vein obstruction . Although the incidence of ophthalmological disorders while on interferon therapy is low, this can result in loss of vision.411 in this prospective case series study, one hundred patients with chronic hepatitis - c under interferon (inf) therapy were consecutively recruited from patients attending the outpatient ophthalmic clinic at the university hospital, faculty of medicine, al - azhar university, assuit . The study protocol was approved by the ethics committee at al - azhar university hospital . Written informed consent was obtained from all patients included in the study, and then in addition to the medical and laboratory examination, all patients were subjected to full ophthalmological assessment to evaluate the retinopathy associated interferon therapy . Inclusion criteria were: (1) seropositivity for anti - hcv antibodies determined using elisa; (2) first use of interferon therapy . Exclusion criteria were: (1) previous interferon therapy; (2) patients with hepatitis b; (3) patients with decompensated liver; (4) patients with renal failure; (5) patients with cardiovascular disorders such as coronary insufficiency, congestive heart failure, and ischemic stroke; (6) patients with a dense cataract, visual field abnormality, glaucoma, or any other ocular abnormalities . Evaluation before treatment: history of visual loss, hazy vision, and ocular disease or ocular surgeries ophthalmological examination: (1) best corrected visual acuity (bcva) measured using landolt s broken rings; (2) pupillary reaction (direct and consensual) and if there is afferent pupillary defect; (3) full slit - lamp examination; (4) intraocular pressure (iop) measured by goldmann s applanation tonometer; (5) fundus biomicroscopy (carried out after maximum pupillary dilation by a combination of tropicamide 1% eye drops and phenylephrine 2.5% eye drops using both slit - lamp biomicroscopy with noncontact + 90 d volk lens and/or goldmann contact lens); (6) examination of the retinal periphery using an indirect ophthalmoscope . Medical examinations and abdominal ultrasonography investigations: (1) ocular, both colored fundus photography and fundus fluorescein angiography (ffa); (2) laboratory (including enzyme - linked immunosorbent assay (ellsa) test for hepatitis surface ag and ab; polymerase chain reaction (pcr) for hepatitis c virus rna; full blood count; prothrombin time; direct and indirect bilirubin; fasting blood sugar; serum creatinine; liver enzymes (serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase); alanine aminotransferase . Patients were treated with pegylated interferon -2a at a dose of 180 mcg injected subcutaneously once weekly for 48 weeks . All patients underwent clinical and laboratory assessments, ophthalmic examination, at 15 days, 1 month, 2 months, 3 months, 6 months, 8 months, and 9 months after the start of ifn treatment . Patients were treated with pegylated interferon -2a at a dose of 180 mcg injected subcutaneously once weekly for 48 weeks . All patients underwent clinical and laboratory assessments, ophthalmic examination, at 15 days, 1 month, 2 months, 3 months, 6 months, 8 months, and 9 months after the start of ifn treatment . Initially, 120 patients fulfilled the criteria for ifn therapy, but 20 of them were excluded due to non - compliance with the follow - up visits and due to the presence of diabetic or hypertensive retinopathy . Also, two patients died during the study time, due to a disease not related to hepatitis, so these patients are not included . Therefore, a total of 100 patients (68 male and 32 female), with chronic hcv were selected . After the start of interferon therapy, 16 out of 100 patients (16%) developed retinopathy which was bilateral in 12 (75%) patients and unilateral in 4 (25%) patients . Retinopathy was initially diagnosed by the appearance of a cotton wool spot in 14 patients . In four of the 14 patients, retinal hemorrhage two of the 16 patients who developed retinopathy were diagnosed by retinal hemorrhage without cotton wool spots . One patient (not diabetic or hypertensive) had cystoid macular edema, this was the only patient who complained of the visual disturbance due to interferon . All patients had bcva of (6/6) during ifn treatment except for three patients: the one who had cystoid macular edema (bcva was 6/12 and returned to 6/6 after cessation of treatment) and two patients with ametropic amblyopia . In addition, we confirmed that all patients with hypertension had no retinopathy using the standard classification . To exclude the possibility of the development of diabetic or hypertension retinopathy after ifn treatment, we deleted the data obtained from patients who had diabetes or hypertension and any retinopathy 2 months after the cessation of ifn treatment . Retinopathy was first diagnosed 214 weeks after the start of treatment and disappeared in 11 of the 16 patients despite the continuation of therapy . A few cotton wool spots, retinal hemorrhages, or both were observed in the remaining five patients at the end of treatment but resolved within 1 month after treatment was stopped . No other ocular complications (ie, cataracts, glaucoma, retinal artery, or vein obstruction) were observed . The mean hemoglobin values, white blood cell, red blood cell, and platelet counts decreased significantly (p <0.0001) during ifn treatment . The clinical profiles and laboratory data of the patients are shown in tables 1 and 2, respectively . Interferon - associated retinopathy was first recognized in 1990 when ikebe and associates reported a 39-year - old patient who developed retinal hemorrhages and cotton wool spots following intravenous administration of interferon.12 the underlying mechanisms of interferon - induced retinopathy are not completely understood . Guyer et al13 had proposed an immune complex deposition causing occlusion of retinal capillaries leading to cotton wool spot formation . On the other hand, an ischemic insult, similar to that seen in hypertensive and diabetic patients could be another potential mechanism by which these changes develop.14 some studies15,16 have shown a relatively higher incidence of retinopathy (symptomatic or asymptomatic) ranging from 19% to 64% during treatment with interferon . Panetta and gilani17 and cuthbertson et al14 show relatively lower incidences of retinopathy (3.8% and 6.25%, respectively). Similar to other studies,4,5,9 our study also shows that retinal changes usually develop within the first 3 months of interferon treatment, a fact that may favor an immune etiology rather than a cumulative effect of interferon leading to the development of retinopathy . Retinopathy developed by week 14 in most of the patients (13/16, 81.25%) after the start of therapy and disappeared in the majority of the patients (11/16, 68.75%) during the 48 week period in which the patients were receiving the treatment . This suggests that treatment can be continued despite the development of retinopathy in many patients . However, two patients who developed cotton wool spots early in the therapy (2 weeks) thereafter suffered from retinal hemorrhage in a prolonged manner . Therefore, patients who develop cotton wool spots early on during therapy should be carefully monitored . However, as reported in previous studies,4,13,14 most of the patients with retinopathy in this study were asymptomatic . This study confirms previous reports that retinopathy is a temporary and asymptomatic complication of interferon therapy . Therefore there is no rule for suspending the use of interferon in patients who develop retinopathy or any other ocular side effects . It s commonly agreed among authors that careful monitoring should be performed in the presence of any ocular sign, even without symptoms.18

Nathan, as an oncology fellow, knew well that white blood cells fought infections . This was an experiment repeated in front of us all the time, he says: chemotherapy lowered his patients' white blood cells and increased their risk of infections . Mackaness had shown that macrophage activation did not depend on direct contact with t cells (1), suggesting the possibility of a secreted factor . When nathan tested the supernatant from activated t cells, he saw that it did indeed induce macrophage activation (2). Nathan got a rough idea of the molecular weight (3), but that was the best anyone could do, he says . Protein separation methods were primitive, and cloned proteins and monoclonal antibodies would only become available a decade later . Henry murray, one of nathan's collaborators, sums up the feeling of frustration: we were all nibbling at the edges of the same problem . Nathan therefore changed tack to take a closer look at the activated macrophages . Short - lived neutrophils were known to produce hydrogen peroxide, and nathan found the same was true of longer - lived activated macrophages (4). Unlike previous signs of macrophage activation increased spreading, phagocytosis, and glucose metabolism this so - called respiratory burst ifn had been on the cover of time magazine, and recombinant murine ifn was found to induce macrophages to kill tumor cells (5). Nathan, now a faculty member in zanvil cohn's macrophage factory at rockefeller university (new york, ny), thought ifn might also activate macrophages to kill intracellular parasites . Consistent with this idea, ifn was made by antigen - stimulated t cells and was associated with defense from infection . Now the respiratory burst gave nathan an assay, berish rubin (down the street at the new york blood center) supplied an ifn monoclonal antibody, and a phone call to genentech yielded recombinant ifn. In a seminal paper published in the journal of experimental medicine in 1983, nathan was thus able to show that depleting ifn from unpurified t cell supernatants decreased the respiratory burst activity and the killing of intracellular protozoa in human macrophages . Adding back recombinant ifn into this mix restored macrophage activation (6). I had an assay, a hunch, a history of purifying proteins that did this, and the serendipity of meeting with people nearby who had the antibody . Nathan next showed that ifn worked in people . Injecting recombinant ifn directly into cutaneous lesions of lepromatous leprosy patients induced macrophage infiltration, hydrogen peroxide production, and killing of the causative pathogen, mycobacterium leprae (7). In the 1990s, the macrophages of children with ifn receptor deficiencies were shown to be defective in killing mycobacteria (8). Tracing the pathway from t cells to macrophages to bacteria started, for nathan, in 1967, and he says we still haven't finished making the molecular links.

Was initiated by our team about a decade ago, and this has been continued at the medical research center, kasturba health society, mumbai . The comparative effects of three plant products on perimenopausal symptoms, quality of life (qol) and clinical biochemistry have been reported by our group earlier . Our observation of frequent mood changes and sleep disturbances in women in the premenopausal group prompted us to undertake a study of cyclic symptoms in maitreyi hcp . In this communication, we describe the preliminary findings from the analysis of premenstrual symptoms in 200 consecutive women who underwent a comprehensive check - up in the maitreyi hcp . Diagnosis of premenstrual tension syndrome (pmts) is based on the following criteria as per the american association of obstetricians and gynecologists: a 30% increase in the intensity of symptoms of premenstrual syndrome (measured using a standardized instrument) from cycle days 510 as compared with the 6-day interval before the onset of menses and documentation of these changes for at least two consecutive cycles . At least one of the following affective and somatic symptoms during the 5 days before menses in previous cycles . Affective symptoms: depression, angry outbursts, irritability, anxiety, confusion, social withdrawal . Somatic symptoms: breast tenderness, abdominal bloating, headache, swelling of extremities; symptoms relieved from days 4 through 13 of the menstrual cycle . To determine the prevalence of premenstrual cyclic symptoms in women who attended the maitreyi hcp . To determine the prevalence of premenstrual cyclic symptoms in women who attended the maitreyi hcp . Maitreyi's hcp for women above 35 includes comprehensive health questionnaire, gynecological and physician's check - up, biochemical investigations of complete blood count, glucose tolerance test, lipid profile, liver functions, serum creatinine and serum thyroid stimulating hormone . Pap smear, pelvic sonography and urine and stool examination for occult blood are also included . A multidisciplinary trans - system team offers this community - oriented programme to women with voluntary services by the consultants . Bone mineral density and mammography this analysis is a preliminary report restricted to the premenstrual cyclic symptomatolgy in 200 consecutive women who attended this program over 2 years . A special case record form has been used and a detailed medical and surgical history is included in the same . Twelve women who continued to menstruate beyond 50, up to 55 years, have been included in the analysis . Only those who had symptoms at least during two consecutive cycles or more were included in the analysis . The american society of obstetricians and gynecologists and the american psychiatric society have now brought out the consensus criteria for inclusion of the condition in pmts or premenstrual dysphoric disorder (pmdd), the latter usually requiring treatment for neuropsychiatric symptoms . Accordingly, pmts is said to be present when the woman reports three or more of the following symptoms in two cycles or more[46]: there are 36 symptoms as listed below in the sf-36 mental component . In the present analysis, the symptoms were classified as per the medical outcomes study short form-36 for assessment of premenstrual symptoms, and the presence of three of these occurring over two or more cycles was labelled as pmts: anxiety, irritability, depression, tension, mood swings, loss of self - control, difficulty in concentration, insomnia, confusion, headache, crying attacks, fatigue, aches, breast tenderness, cramps, bloatedness, food craving, visual disturbances, tiredness, fatigue, puffiness of the face, weight gain, altered libido, aggression, nausea, vomiting, acne, skin rashes, constipation, diarrhea, joint stiffness, backache, abdominal cramps / pain, anger attacks, aggravation of epilepsy, aggravation of asthma . Of 107 women, 26 (24.3%) did not report any premenstrual symptoms at all and 81 (75.7%) reported at least one symptom [table 1]. Number of symptoms experienced by women in different age groups in two or more cycles maximum frequency was seen in women from 35 to 50 years of age . Forty women (37.4%) who reported premenstrual symptoms had three or more cyclic symptoms for more than two cycles and could be classified as those with pmts . Fifteen (14%) had five or more symptoms cyclically in at least two cycles . Almost 10% reported five or more symptoms inclusive of anger attacks and could be labelled as pmdd . Mastalgia was the most common symptom (50.5%), followed by mood changes (46.7%), depression (7.5%) and anger attacks (6.5%). Pmdd was present in 10% of the cases who were referred to a clinical psychologist or a psychiatrist . Many women have premenstrual cyclic symptoms of psychological or physical nature and, sometimes, these limit their functional capacity . The symptoms due to neurohormonal imbalance or ageing may start as early as 35 years of age . Pmts is an entity that causes considerable morbidity, and in about 3% of the women, it may disrupt the woman's life when severe symptoms recur . When there are severe symptoms like anger attacks, depression and suicidal thoughts, special attention including psychiatric consultation may be required, i.e. The pmdd . The prevalence of pmts in general is about 210% for disabling symptoms, while minor symptoms may be present in up to 85% of the women. [468] in the present series, 75.7% reported at least one symptom cyclically . In the present series halbreich has reported that in about 20% of the women, the symptoms are severe enough to warrant treatment . Dean et al . Have reported a prevalence of 1930% when all women from 18 to 45 years of age were screened . In a recent survey of 3,913 women of 15 - 54 years of age, tschudin et al . Observed that about 10% reported pmts and 3.1% reported pmdd . When the indian scenario is considered, chaturvedi et al . In the 1990s have reported a prevalence of 20% in a general population and severe symptoms in 8% . The same authors have, in a later study, reported suicidal ideas and/or death wish during the premenstrual period in 10% of the subjects, more so among college students and industrial working women as compared with housewives . Banerjee et al ., in a group of 62 women, reported pmdd in 6.4% . There is a close association between mood disorders and the sleep rhythm and disturbances in sleep pattern in premenopausal age. [31314] many women and their physicians regard pmts as a physiological phenomenon and do not think treatment is necessary . However, it does affect qol in some and it is therefore essential to identify which women will benefit from lifestyle management and which will require pharmacological intervention . Those who want to reach their full functional potential and who find that the symptoms are hampering their progress or interfering with normal daily routine should be certainly offered safe pharmacological interventions provided that they come for routine follow - ups . Because there are many etiological factors that have been described as probably causative, and multiple causes may be present in an individual woman, there is a wide variety of treatment options and combinations depending on specific symptoms and their severity . The postulated causes range from hormonal imbalances like progesterone deficiency, prolactin excess, thyroid hypofunction, fluctuation in circulating level of estrogen; electrolyte disturbances like rennin - angiotensin alterations, antidiuretic hormone excess, decreased colloidosmotic pressure; neurotransmitter disturbances with serotonin or gamma - amino - butyric - acid, or -endorphin activity alternations, serotonin metabolism alternations or prostaglandin imbalance; cytokine imbalances like excess of interleukin (il)-6, il-1 and tumor necrosis factor (tnf)-; nutritional deficiencies like vitamin b or vitamin d deficiency; lack of exercise or psychosocial disturbances. [8101315] the treatment therefore will depend on the particular symptom complex in the individual woman . In concordance with other authors, lifestyle measures like regular exercise, balanced nutritious diet and regular hours of sleep will benefit all, and may be adequate for some. [1921] for others, pharmacological interventions may be helpful if required . The therapy is multipronged and has to be individualized . While simple measures like the institution of physical exercises, relaxation techniques like yoga and vitamin and mineral supplements are adequate for most women,[1924] some require hormonal therapy with combined oral contraceptives or regulation of fluid and electrolytes . As a matter of fact, combined oral contraceptives are used frequently in the more severe cases and, drosperidone, containing newer combinations, is relatively free from side - effects . Selective estrogen modulators, including nonsteroidal agents like centchroman, also could be useful, particularly in women with contraindications to hormones . Few women need psychiatric consultation and will benefit from antidepressants, particularly sertraline or fluoxetine, while some respond to prolactin inhibitors like bromocriptine or cabergoline . Complementary and alternative therapies like soy, hypericum perforatum and gingko biloba have a definite role to play. [242931] evening primrose oil does not have significant efficacy . We have observed relief of mild to moderate premenstrual symptoms with soy, gycyrrhiza glabra and withania somnifera . We have also observed complete relief of pmts in two women treated with an extract rich in saraca asoka, prescribed for menorrhagia, which was also controlled . There is need for more research in this area because commonly used drugs may have side - effects that may prevent regular long - term use [table 2]. Common side - effects of existing pharmacologic therapies for pmts recently, two well - designed studies have shown that pmts leads to loss of work hours and economic losses, and does not allow women to reach their full potential. [3436] effective and safe therapy is indicated . It is also important to bear in mind that other chronic diseases like migraine or depression or irritable bowel syndrome could be exaggerated during the premenstrual phase, and a multidisciplinary team should evaluate and manage such cases . Many women and their physicians regard pmts as a physiological phenomenon and do not think treatment is necessary . However, it does affect qol in some and it is therefore essential to identify which women will benefit from lifestyle management and which will require pharmacological intervention . Those who want to reach their full functional potential and who find that the symptoms are hampering their progress or interfering with normal daily routine should be certainly offered safe pharmacological interventions provided that they come for routine follow - ups . Because there are many etiological factors that have been described as probably causative, and multiple causes may be present in an individual woman, there is a wide variety of treatment options and combinations depending on specific symptoms and their severity . The postulated causes range from hormonal imbalances like progesterone deficiency, prolactin excess, thyroid hypofunction, fluctuation in circulating level of estrogen; electrolyte disturbances like rennin - angiotensin alterations, antidiuretic hormone excess, decreased colloidosmotic pressure; neurotransmitter disturbances with serotonin or gamma - amino - butyric - acid, or -endorphin activity alternations, serotonin metabolism alternations or prostaglandin imbalance; cytokine imbalances like excess of interleukin (il)-6, il-1 and tumor necrosis factor (tnf)-; nutritional deficiencies like vitamin b or vitamin d deficiency; lack of exercise or psychosocial disturbances. [8101315] the treatment therefore will depend on the particular symptom complex in the individual woman . In concordance with other authors, lifestyle measures like regular exercise, balanced nutritious diet and regular hours of sleep will benefit all, and may be adequate for some. [1921] for others, pharmacological interventions may be helpful if required . The therapy is multipronged and has to be individualized . While simple measures like the institution of physical exercises, relaxation techniques like yoga and vitamin and mineral supplements are adequate for most women,[1924] some require hormonal therapy with combined oral contraceptives or regulation of fluid and electrolytes . As a matter of fact, combined oral contraceptives are used frequently in the more severe cases and, drosperidone, containing newer combinations, is relatively free from side - effects . Selective estrogen modulators, including nonsteroidal agents like centchroman, also could be useful, particularly in women with contraindications to hormones . Few women need psychiatric consultation and will benefit from antidepressants, particularly sertraline or fluoxetine, while some respond to prolactin inhibitors like bromocriptine or cabergoline . Complementary and alternative therapies like soy, hypericum perforatum and gingko biloba have a definite role to play. [242931] evening primrose oil does not have significant efficacy . We have observed relief of mild to moderate premenstrual symptoms with soy, gycyrrhiza glabra and withania somnifera . We have also observed complete relief of pmts in two women treated with an extract rich in saraca asoka, prescribed for menorrhagia, which was also controlled . There is need for more research in this area because commonly used drugs may have side - effects that may prevent regular long - term use [table 2]. Recently, two well - designed studies have shown that pmts leads to loss of work hours and economic losses, and does not allow women to reach their full potential. [3436] effective and safe therapy is indicated . It is also important to bear in mind that other chronic diseases like migraine or depression or irritable bowel syndrome could be exaggerated during the premenstrual phase, and a multidisciplinary team should evaluate and manage such cases . It is very important to ensure that the first option is nonpharmacological as many women may respond to it . Pharmacotherapy can be added in recalcitrant cases and must be individualized as per the woman . Great care should be taken to avoid side - effects and drug interactions because multiple therapies may be used . Medicinal plants may be a safe alternative in some women . Because pmts could be a predictor for menopausal syndrome, it will be useful to warn women with pmts and their families of a flare up or the need for long - term follow - up . Have reported pmts to be predictive of menopausal syndrome in a long - term follow - up study and have shown the association of hormonal imbalance with depressive symptoms in women.

According to the korean national statistical office, the main cause of death in korea was cancer in 2012 . The total number of deaths due to cancer in 2012 was 267,221, which was increased by 9,825 (3.8%) compared to 2011.1 this was the all - time high since 1983 . Currently, the treatment of cancer patients in the medical field consists mainly of surgery, radiation therapy, and chemotherapy . Seventy to eighty percent of cancer patients in chemotherapy experienced side effects such as anemia, the decreased number of white blood cells and/or platelets, oral mucositis, vomiting, diarrhea, hair loss, and disabled generative functions as chemotherapy also affects the rapidly proliferating normal cells.2 most side effects are temporary and patients recover fully; but, some side effects may take months or years to completely disappear . To decrease side effects from non - specific cytotoxicity, new drugs, such as targeted agents, are being developed to selectively destroy specific cancer cells . However, because of problems, such as tolerance manifestation, partly critical toxicity, decrease in sensitivity, and restriction in subjects, new drugs take only a small portion of the cancer treatment realm.3 chemoprevention is defined as the use of nontoxic or relatively safe chemical substances, such as vitamins, plant extracts, and pharmaceuticals to prevent cancer . It is effective in decreasing cancer risk and is helpful in early stages of cancer.4 hence, regular consumption of anticancer foods that display no or little toxicity to the body and boost efficacy of anti - tumor drugs or stop the development of tolerance would be beneficial in preventing cancer . Thus, consumption of plant - derived natural foods to prevent cancer with few side effects and toxicity is crucial.5 recently, there has been a growing interest in probiotics due to the discovery of multidrug - resistant organisms that are tolerant to antibiotics . Probiotics is derived from a greek word meaning for life and holds the opposite meaning from antibiotics which means against life. Probiotics consist of microorganisms and substances, which modify the intestinal microflora, and when used as dietary supplement they have positive effects on health such as fortification of host immune function, suppression of diarrhea, and inhibition of carcinogenesis.6 kimchi, a korean traditional food, is fermented by probiotic lactic acid bacteria.7 kimchi lactic acid bacteria are known to suppress the activity of carcinogen - activating enzymes, such as azoreductase, nitroreductase, 7--dehydrogenase, -glucosidase, and -glucuronidase and inactivate or neutralize the cancer - causing agents and pathogenic microbes.8 in addition, the intake of lactic acid bacteria as part of kimchi improves bowel movement, strengthens immunity, ameliorates hepatocirrhosis, and decreases serum cholesterol levels.9,10 among lactic acid bacteria identified during the fermentation of kimchi, weissella, lactobacillus, leuconostoc and pediococcus species are known to play an important role in kimchi fermentation.1113 this commentary deals with anticarcinogenic effects of kimchi and lactic acid bacteria, weissella cibaria and lactobacillus plantarum separated from kimchi, which are known to be predominant in kimchi . Kimchi, a fermented cabbage indispensable to korean cuisine, was once considered to be a risk factor for stomach cancer . However, unless one consumes kimchi that contains excess salt, kimchi, in general, is a healthy food recognized for its antioxidant, antiobese, cancer preventive, and other health beneficial effects.7 cancer preventive / anticarcinogenic activity of kimchi is associated with the type of ingredients and products formed during fermentation.14 the kimchi s main ingredient, chinese cabbage, has been considered to be effective in preventing stomach cancer according to epidemiologic studies and is known to inhibit colorectal carcinogenesis due to its abundance in dietary fiber.15 garlic, in particular, has been acknowledged for its anticancer effect due to high contents of organosulfur compounds (11 to 35 mg / g). Thirty - three types of organosulfur compounds have been identified in garlic.16 organosulfur compounds in garlic have been shown to facilitate the detoxification of carcinogens by glutathione - s - transferase, modulate the activity of metabolizing enzymes such as cytochrome p450s, and inhibit the formation of dna adducts in several target tissues.1719 antiproliferative activity of organosulfur compounds has been demonstrated in tumor cell lines including colon, prostate, and breast, which is mediated by induction of apoptosis via the caspase-3 signaling pathway and cell - cycle arrest.20,21 one possible mechanism of cancer preventive effects of garlic in stomach cancer development involves its antimicrobial activities against helicobacter pylori, a major risk factor of stomach cancer.22 hot red chili pepper powder, one of the main ingredient of kimchi, contains capsaicin (trans-8-methyl - n - vanillyl-6-nonenamide). Although the role of capsaicin in carcinogenic processes has been controversial, there is accumulated evidence that capsaicin is capable to induce apoptosis of cancer cells by generating excess reactive oxygen species.23 thoennissen et al.24 demonstrated that capsaicin caused cell - cycle arrest and apoptosis in breast cancer cells by modulating the egfr / her-2 pathway and inhibited the development of pre - neoplastic breast lesions by up to 80% without toxicity . In addition to each ingredient of kimchi, kimchi extract was used to investigate cancer preventive / anticarcinogenic activity of kimchi . Extract of kimchi fermented for 3 weeks inhibited the proliferation of human cancer cell lines (gastric adenocarcinoma and acute promyelocytic leukemia). Leukemia cells treated with kimchi extract showed increased apoptosis and decreased mitochondrial transmembrane potential.25 the juice of 3-week fermented kimchi suppressed the growth of k-562 human leukemia cells and mg-63 human osteosarcoma cells but no toxicity of kimchi juice was found in normal cells.26 sarcoma180 cells transplanted mice were treated with methanol extract of 3-week fermented kimchi . Animals provided kimchi extract showed in smaller tumor weight and decreased malondialdehyde formation compared to those of the control group.27 although there has been no general acceptance on how to define probiotic microorganisms, lactobacillus species, bifidobacterium sp, saccharomyces boulardii, and some other microbes have been thought and used as probiotic strains.28 these probiotic microorganisms have been used as food supplement in order to achieve health benefit effects of them . There have been many studies reported on the functionality of lactic acid bacteria in the fermentation process of kimchi and their importance in the health beneficial effects of kimchi, including prevention of cancer.29 according to pyrosequencing analysis of commercial kimchi samples to identify kimchi lactic acid bacteria, the genus weissella was predominant at 44.4% . W. koreensis was 27.2% and w. cibaria was 8.7%.7 weissella is a newly separated lactic acid bacteria of lactobacillus family that was identified by a recent dna technique . It is a gram - positive and catalase negative bacteria included in geneally recognized as safe.30 the main lactic acid bacteria involved in kimchi fermentation has long been known as lactobacillus genus and leuconostoc genus, but more recently, weissella genus lactic acid bacteria has been newly separately identified.30 weissella is an early dominant in kimchi fermentation and produces lactic acid, acetic acid, alcohol, dextran, and co2, which account for the unique fresh taste and texture of kimchi.31,32 weissella species found in kimchi are w. cibaria, w. koreensis, and w. hanii.12 sources of each weissella species are shown in table 1.33 w. cibaria, newly named by bjorkroth and others, has been separated from korean kimchi and various other sources, including fermented foods, greek salami, spanish sausages, and animal and human excrements . W. cibaria is gram - positive, non - pore formulating, non - motile, hetero lactic acid fermented, and catalase negative bacillus, and cannot produce dextran from sucrose.34,35 studies have reported that microorganisms, including lactic acid bacteria, secreted exopolysaccharides (eps) with the anticancer, anti - inflammatory, immune modulating, and blood cholesterol declining functions.36 it was demonstrated that w. cibaria, leuconostoc mesenteroides and w. confusa are able to produce eps . Among these lactic acid bacteria, w. cibaria exhibited higher production of eps which indicates more acid resistance (table 2).37 w. cibaria was reported to have anticancer activity, immune modulating activity, anti - inflammatory activity, and antioxidant activity . Based on the patent related to the w. cibaria s anticancer activity registered by cha et al.,38 the anticancer activity in w. cibaria w. cibaria bacteria was incubated for 24 hours in deman rogosa sharpe agar badge, diluted with phosphate buffered saline, and 10% concentration of bacteria samples were provided to normal cell strains and colorectal cancer cell strains for 72 hours . Cell growth was suppressed by treatment of w. cibaria in colorectal cancer cells but not in normal cells . Ahn et al.33 presented that the immune control effect of w. cibaria was stronger than the well - known probiotic bacterium, l. rhammosus gg (lgg). W. cibaria produced higher levels of nitric oxide, nuclear factor (nf)- b, cytokines (e.g., interleukin-1 and tumor necrosis factor-) than lgg, suggesting that w. cibaria is more effective in immune control compared to lgg . Furthermore, w. cibaria is known to have an antiviral function against the avian influenza virus.39 ornithine, a type of amino acid produced from its arginine precursor, is reported to accelerate growth hormone excretion and has an antiobesity effect . During the kimchi fermentation process, the level of arginine, an ornithine precursor, has decreased while the ornithine level increased rapidly, which suggested that kimchi is a source of ornithine . Moreover, a correlation between the amount of w. cibaria and the amount of ornithine in kimchi was observed.40 use of w. cibaria as a starter for food fermentation promoted the formation of ornithine from arginine, which in turn provide health beneficial effects, such as antiobesity due to high levels of ornithine in fermented food.41,42 lactobacillus genus is a microorganism which does not form spores and is an anaerobic and facultative anaerobic gram - positive bacterium . This bacterium is not only widely dispersed in nature but is also found in human oral cavity and digestive organs . This bacterium is a beneficial microorganism that is widely used as a starter for various fermented dairy products.43 the main kimchi lactic acid bacteria of lactobacillus is l. plantarum . The rapid increase of l. plantarum in the late stage of kimchi fermentation produces large amounts of organic acid, which is known as the main substance that acidifies kimchi . Acid tolerance of l. plantarum allows us to use this bacterium for natural antibacterial and antifungal products . L. plantarum s cancer preventive potential has been tested by the ames mutation assay and the sos chromotest . When cabbage was brined with 3% salt concentration and kimchi was fermented at 5c for 3 weeks, kimchi extract exerted an antimutagenic effect against aflatoxin b1.44 l. plantarum, isolated from kimchi, also exhibited a strong antimutagenic effect against n - methyl - n-nitro - n - nitrosoguanidine, 4-nitroquinoline-1-oxide.45 furthermore, l. plantarum, isolated from kimchi, had stronger antimutagenic effects compared to lactic acid bacteria originated from fermented milk.46 the macrophagocytes provided with l. plantarum separated from kimchi strengthened phagocytosis, and displayed anticancer effects in aseites carcinoma and solid tumor due to the polysaccharide chains of muramic acid in the l. plantarum s cell well . Among lactic acid bacteria s cell wall substances, polysaccharide types rather than glycopeptide play a pivotal role in cancer suppression.47 to evaluate the possible use of l. plantarum as a probiotic, lee et al.48 measured the survival rate in artificial gastric fluid and intestinal fluid, adhesion compatability with human intestinal caco-2 cells, and antibacterial activity . As a result, l. plantarum strains showed more than 90% survival rate in artificial gastric fluid and intestinal fluid, and more than 400 times the survival rate of the control strain, l. casei . When pathogenic bacteria and lactic acid bacteria were incubated together, l. plantarum suppressed the adhesion of intestinal crypt cells with the pathogenic bacteria, and also suppressed pathogenic bacteria growth of escherichia coli, stapylococcus aureus, and salmonella typhimurium by 93.4%, 75.9%, and 69.3% respectively . Weissella is a newly separated lactic acid bacteria of lactobacillus family that was identified by a recent dna technique . It is a gram - positive and catalase negative bacteria included in geneally recognized as safe.30 the main lactic acid bacteria involved in kimchi fermentation has long been known as lactobacillus genus and leuconostoc genus, but more recently, weissella genus lactic acid bacteria has been newly separately identified.30 weissella is an early dominant in kimchi fermentation and produces lactic acid, acetic acid, alcohol, dextran, and co2, which account for the unique fresh taste and texture of kimchi.31,32 weissella species found in kimchi are w. cibaria, w. koreensis, and w. hanii.12 sources of each weissella species are shown in table 1.33 w. cibaria, newly named by bjorkroth and others, has been separated from korean kimchi and various other sources, including fermented foods, greek salami, spanish sausages, and animal and human excrements . W. cibaria is gram - positive, non - pore formulating, non - motile, hetero lactic acid fermented, and catalase negative bacillus, and cannot produce dextran from sucrose.34,35 studies have reported that microorganisms, including lactic acid bacteria, secreted exopolysaccharides (eps) with the anticancer, anti - inflammatory, immune modulating, and blood cholesterol declining functions.36 it was demonstrated that w. cibaria, leuconostoc mesenteroides and w. confusa are able to produce eps . Among these lactic acid bacteria, w. cibaria exhibited higher production of eps which indicates more acid resistance (table 2).37 w. cibaria was reported to have anticancer activity, immune modulating activity, anti - inflammatory activity, and antioxidant activity . Based on the patent related to the w. cibaria s anticancer activity registered by cha et al.,38 the anticancer activity in w. cibaria was tested with colorectal cancer cells . W. cibaria bacteria was incubated for 24 hours in deman rogosa sharpe agar badge, diluted with phosphate buffered saline, and 10% concentration of bacteria samples were provided to normal cell strains and colorectal cancer cell strains for 72 hours . Cell growth was suppressed by treatment of w. cibaria in colorectal cancer cells but not in normal cells . Ahn et al.33 presented that the immune control effect of w. cibaria was stronger than the well - known probiotic bacterium, l. rhammosus gg (lgg). W. cibaria produced higher levels of nitric oxide, nuclear factor (nf)- b, cytokines (e.g., interleukin-1 and tumor necrosis factor-) than lgg, suggesting that w. cibaria is more effective in immune control compared to lgg . Furthermore, w. cibaria is known to have an antiviral function against the avian influenza virus.39 ornithine, a type of amino acid produced from its arginine precursor, is reported to accelerate growth hormone excretion and has an antiobesity effect . During the kimchi fermentation process, the level of arginine, an ornithine precursor, has decreased while the ornithine level increased rapidly, which suggested that kimchi is a source of ornithine . Moreover, a correlation between the amount of w. cibaria and the amount of ornithine in kimchi was observed.40 use of w. cibaria as a starter for food fermentation promoted the formation of ornithine from arginine, which in turn provide health beneficial effects, such as antiobesity due to high levels of ornithine in fermented food.41,42 lactobacillus genus is a microorganism which does not form spores and is an anaerobic and facultative anaerobic gram - positive bacterium . This bacterium is not only widely dispersed in nature but is also found in human oral cavity and digestive organs . This bacterium is a beneficial microorganism that is widely used as a starter for various fermented dairy products.43 the main kimchi lactic acid bacteria of lactobacillus is l. plantarum . The rapid increase of l. plantarum in the late stage of kimchi fermentation produces large amounts of organic acid, which is known as the main substance that acidifies kimchi . Acid tolerance of l. plantarum allows us to use this bacterium for natural antibacterial and antifungal products . L. plantarum s cancer preventive potential has been tested by the ames mutation assay and the sos chromotest . When cabbage was brined with 3% salt concentration and kimchi was fermented at 5c for 3 weeks, kimchi extract exerted an antimutagenic effect against aflatoxin b1.44 l. plantarum, isolated from kimchi, also exhibited a strong antimutagenic effect against n - methyl - n-nitro - n - nitrosoguanidine, 4-nitroquinoline-1-oxide.45 furthermore, l. plantarum, isolated from kimchi, had stronger antimutagenic effects compared to lactic acid bacteria originated from fermented milk.46 the macrophagocytes provided with l. plantarum separated from kimchi strengthened phagocytosis, and displayed anticancer effects in aseites carcinoma and solid tumor due to the polysaccharide chains of muramic acid in the l. plantarum s cell well . Among lactic acid bacteria s cell wall substances, polysaccharide types rather than glycopeptide play a pivotal role in cancer suppression.47 to evaluate the possible use of l. plantarum as a probiotic, lee et al.48 measured the survival rate in artificial gastric fluid and intestinal fluid, adhesion compatability with human intestinal caco-2 cells, and antibacterial activity . As a result, l. plantarum strains showed more than 90% survival rate in artificial gastric fluid and intestinal fluid, and more than 400 times the survival rate of the control strain, l. casei . When pathogenic bacteria and lactic acid bacteria were incubated together, l. plantarum suppressed the adhesion of intestinal crypt cells with the pathogenic bacteria, and also suppressed pathogenic bacteria growth of escherichia coli, stapylococcus aureus, and salmonella typhimurium by 93.4%, 75.9%, and 69.3% respectively . This paper describes the cancer preventive / anti - carcinogenic potential of kimchi lactic acid bacteria, focusing on w. cibaria and l. plantarum . The antibiotic tolerant probiotic presented in kimchi suppresses expression of carcinogen activating enzymes and possesses many other health benefits such as suppression of growth and development of pathogenic bacteria, intestinal regulation, and immune boosts . Especially, the w. cibaria and l. plantarum found in kimchi have many effects, like anti - inflammatory, immune modulating, and blood cholesterol reducing activity, which may account for their cancer preventive / anticancer potential . These probiotic lactic acid bacteria in kimchi cannot only be used as an antimicrobials in foods but also be implemented in developing functional foods to reduce the risk of colon cancer.

A glomangioma is a benign vascular tumor derived from the glomus body, a specialized neuromyoarterial structure involved in thermal regulation . It is a subtype of the more generalized category of glomus tumors and should not be confused with the head and neck paragangliomas, such as the glomus tympanicum or glomus jugulare . It should also be distinguished from the glomangiopericytoma, the sinonasal type hemangiopericytoma, which displays different ultrastructural and histochemical characteristics.1 glomangiomas are most commonly found subungually and are exceedingly rare in the head and neck.2 they account for only 0.6% of all nonepithelial tumors of the nasal cavity, nasopharynx, and paranasal sinuses.3 glomangiomas that induce paraneoplastic osteomalacia are even more uncommon, with only one case reported to date.4 while there are several documented cases of oncogenic osteomalacia (oo) caused by glomangiopericytomas,5 we present the second reported case of glomangioma - induced osteomalacia and the first case documented in english . A 42-year - old man with a history of unexplained hip, rib, scapula, and metatarsal fractures; left foot pain with exercise; and weakness presented to our clinic for evaluation of a paranasal sinus tumor . The endocrinology service initially diagnosed him with tumor - induced osteomalacia caused by fibroblast growth factor 23 (fgf23), which prompted an octreotide scan revealing a questionable area of enhancement in the pituitary . A subsequent magnetic resonance imaging (mri) showed an ethmoid mass extending through the cribriform intracranially, measuring 3.9 1.9 2.4 cm at its largest dimensions (fig . The patient reported occasional sinus congestion, decreased sense of smell during the preceding 2 to 3 years and occasional blurry vision in the morning . His laboratory workup revealed a significantly decreased 1,25-vitamin d level of less than 8 ng / ml, a decreased phosphate level to 1.6 mg / dl, a low to normal calcium level of 8.7 mg / dl, and a significantly elevated alkaline phosphatase level at 65.3 g / l . Preoperative mri, t1 with contrast, showing an ethmoid mass extending through the cribriform intracranially . The patient underwent a combined endoscopic endonasal approach to the anterior skull base with tumor resection . The cribriform defect and intraoperative cerebrospinal fluid (csf) leak pathology revealed a vascular neoplasm with a uniform cluster of ovoid cells arranged around the vessels and moderate focal nuclear enlargement (fig . Tumor cells stained positive for cd31 and smooth muscle actin (sma) but negative for cd34, s100, and pan - cytokeratin on immunohistochemistry (fig . The patient reported near - complete resolution of bone pain and improvement in smell, and he had normalization of phosphate, alkaline phosphatase, vitamin d, and other laboratory values . Repeat mri showed gross total resection of the mass and no detectable recurrence (fig . 4). Postoperative mri, t1 with contrast, showing gross total resection of the mass with nasoseptal flap reconstruction of the skull base . Osteomalacia is a disease of the bone characterized by defective mineralization of osteoid from decreased levels of available phosphate and calcium or increased bone resorption . It often presents with diffuse joint and bone pain, easy fracturing, difficulty walking, weakness, and other nonspecific symptoms . Oo is a rare, disabling, and curable form of osteomalacia that affects both sexes equally and usually presents around 40 years of age.6 it is not well described in glomangiomas but has been detailed several times in relation to glomangiopericytomas and other soft tissue and bone tumors, with more than 300 reported cases7 since its debut in 1947.8 it predominantly occurs in the context of mesenchymal tumors and is thought to be due to neoplastic overexpression of fgf23 . This protein inactivates the sodium - phosphate pump in the proximal tubule (prohibiting phosphate reabsorption and inducing renal phosphate wasting) and reduces 1-hydroxylation of 25-hydroxy vitamin d.9 accordingly, common oo laboratory abnormalities include hypophosphatemia, normal or decreased calcium, decreased 1,25-dihydroxy vitamin d3 with resistance to vitamin d supplementation, and elevated alkaline phosphatase, which our patient exhibited . Tumors causing oo tend to be small, occult, and slow growing, making diagnosis remarkably difficult . When other causes of osteomalacia have been ruled out and oo is suspected, clinicians should consider measuring a serum fgf23 level . Elevated fgf23 in this setting should then prompt full body imaging (including hands and feet) to expose the lesion . The current imaging standard for investigation of oo is whole - body mri short tau inversion recovery (stir).10 another commonly used modality is octreotide scintigraphy (octreoscan), which utilizes a radiotracer that binds to somatostatin receptors overexpressed on the causative tumor.11 once the neoplasm is localized, surgical resection with wide margins is the definitive treatment . It is curative in essentially all cases and typically leads to rapid normalization of laboratory values and reversal of most clinical symptoms . The nonspecific presentation of osteomalacia, the obscure nature of the tumors that cause oo, and the rarity of glomangiomas in the head and neck make this patient's diagnosis challenging . We believe this to be the second reported case of paranasal sinus glomangioma - induced osteomalacia and the first reported in english . Many head and neck surgeons may not be aware of its existence or consider it in their differential diagnoses of sinus tumors . This report serves to increase the awareness of this uncommon pathology so that it may be considered and treated in future cases.

Gestational trophoblastic disease describes a number of gynecologic tumors that originate in trophoblastic layer including hydatidiform mole (complete or partial), invasive mole, choriocarcinoma, placental site trophoblastic tumor and epitheloid trophoblastic tumor . Invasive mole may arise from any pregnancy event although in most cases is diagnosed after molar pregnancy . Overall cure rate in low risk patients is nearly 100% and in high - risk patient 90% . In rare cases, molar tissue traverses thickness of myometrium and leads to perforation and acute abdomen and invasive mole infrequently metastasis . The best treatment option is chemotherapy (according to stage and score with single or multiple agent) and in patients that fertility is not the matter, hysterectomy can be done . A 41 years old g3p2ab1 woman referred to firouzgar hospital 2 months after curettage of molar pregnancy with vaginal bleeding and acute abdomen . In workup, hcg 224000 miu / ml and evidence of metastasis was detected . Chemotherapy due to stage 3 and score 9 and surgery due to acute abdomen was done . This case reported about ovarian metastasis and uterine rupture with acute abdomen and involvement of omentum in metastatic invasive mole . Management of this patient was successful . In follow up, she was free of disease without sequel of any kind for five years now . Invasive mole is penetration of molar tissue (complete or partial mole) into myometrium or uterine vasculature (1, 16, 19). Pathologists mention the existence of villi in trophoblastic tissue (8, 12). Locally invasive gestational trophoblastic neoplasia develops in 15% of patients and metastatic form in 4% of patients after evacuation of complete mole and infrequently after partial mole (2). Hcg level (> 100000 miu / ml), excessive uterine enlargement, theca lutein cyst size 6 cm are considered as high risks for developing post molar tumors (high risk mole) (3). The most common symptom of invasive mole is persistent vaginal bleeding after evacuation of molar pregnancy (sub involution of uterus and persistent theca lutein cyst is another symptom). The rise in hcg titer is a laboratory test for diagnosis of invasive mole in follow up of molar pregnancy . Although definite diagnosis of invasive mole is based on pathology (8), with hcg or radiologic diagnosis (9), invasive mole is diagnosed as well . In rare cases, metastasis occurred and the most common sites were lungs (80%) (4), vagina (30%), pelvis (20%), liver (10%), brain (10%), bowel and kidney and spleen (<5%) were other metastatic sites . Metastasis can occur from direct extension of another pelvic neoplasm or by hematogenous or lymphatic spread or by transcelomic dissemination . Possibility of metastasis to ovary is extremely rare which is even much lower than that of nongestational primary ovary choriocarcinoma with an incidence of 1 in 3.710 (5). Invasive mole is curable with chemotherapy but hysterectomy decreases the need for multiple courses of chemotherapy and in patients with heavy bleeding or sepsis for control of complication and stabilization, chemotherapy is needed (6, 11). Here a case with invasive mole after evacuation of molar pregnancy, presented with acute abdomen and in surgery metastasis to ovary and omentum and parametrium was detected . A 41- year- old housewife woman, gravid 3 para 2 live child 2 (all nvd) with a last 10 years old child referred to firouzgar hospital on 24 august 2010 with a history of a dilation and curettage and 2 months of cough . On examination, she was febrile (38c ., tachycardia, leukocytosis (16000) and uterus was tender and enlarged . Vaginal bleeding persisted and second curettage was done in another hospital . At that time, chest x - ray was normal . In our hospital, report of sample pathology given from first and second curettage was complete mole . After molar evacuation, the patient was not monitored for weekly determination of hcg level . She received antibiotics (due to fever) and work up for malignant trophoblastic disease or persistent gtn was done because of high hcg level (224000 miu / ml) and much too enlarged uterus and involvement of the whole uterine parenchyma, which was detected on sonography (largest tumor size in uterus was 6 cm). Thyroid function test demonstrated hyperthyroidism . In the metastatic work up, ct scan of chest and abdomen and brain was carried out . Ct scan of the abdomen and pelvis showed involvement of the left adnexa and pelvic lymphadenopathy . The patient s chest ct scan showed multiple bilateral round pulmonary metastatic lesions that were 2 cm in diameter, illustrated in figure 1 . Pulmonary metastasis in invasive mole once the disease was diagnosed, the patient chemotherapy with ema started (table 1). Because of acute abdomen and severe abdominal pain and unstable vital signs with diagnosis of perforative peritonitis, the patient was taken up for emergency laparotomy . Due to hyperthyroid state, in induction of anesthesia, blocker agent was administered . In this patient, subtotal hysterectomy and left salpingo oophorectomy and resection of omentum were done . In gross uterus, uterine wall was perforated by tumoral invasion on the left side of posterior wall (figure 2). Enlarged uterus, ovarian metastasis, perforation of uterus, and myometrium invasion the patient was iii:9 according to figo staging and who scoring (7) the bilateral uterine parameters, the external surface of the endocervical canal and the omentum were invaded by tumors . Villous formation in myometrium villosity with necrosis in invasive mole this patient was treated with 4 courses of chemotherapy with ema - co . After negative hcg (<5), she received an additional 3 courses of chemotherapy to reduce the relapse . Hcg titer was later tested monthly for1 year . When the first year of testing hcg terminated, hcg testing on a bimonthly basis for 2 years was done . The patient was followed up to 5 years and after that the patient was free of tumor and the hcg was negative . Invasive mole with rupture of uterus and metastasis to ovary and omentum and manifestation of acute abdomen was rare (5). According to epidemiological retrospective survey, invasive mole secondary to hydatidiform mole occurred in six months after evacuation (5, 3). Partial and complete hydatidiform mole are distinct disease processes with characteristic cytogenetic, histologic, and clinical features (8). (13) reported a case of invasive mole presenting as acute hemoperitoneum that was similar to the present case . Molar tissue can penetrate myometrium (9) and lead to uterine perforation (13) and cause vaginal bleeding due to erosion of uterine vessels (14, 15). Similar to atala et al.s case report in 1991(14), in our case, uterine perforation with acute intra peritoneal hemorrhage was seen . Due to ease of entry of molar tissue into large venous lake present in myometrium and pelvis in pregnancy or trophoblastic disease metastasis of invasive mole commonly in lungs and after lungs in vagina, cervix, broad ligament (16, 19), in our case metastasis to lung and ovary and omentum although theca lutein cysts in high - risk mole due to high serum hcg level are seen normally, they regress spontaneously within 24 months (17). Non gestational choriocarcinoma of ovary is differential diagnosis (5) but due to occurrence of metastasis to ovary in our case after molar pregnancy, this differential diagnosis was not a matter . As in a report of invasive mole in fallopian tube (21), the first misdiagnosis of our case in another hospital, repeat curettage was done, although the first diagnosis could be persistent gtn . In another case report (15) from bruner, similarity with our case was seen . Combination chemotherapy should be continued in such cases as far as toxicity permits until the patient achieves 3 consecutive normal hcg levels (9, 18). As normal hcg levels are attained, 3 additional courses of chemotherapy are administered to reduce the risk of relapse . In low risk patients, one or two courses are adequate (19). In our case, 3 courses of chemotherapy after normalization of hcg were done . Hysterectomy may be required in invasive mole in order to control vaginal bleeding and in unstable patient or in sepsis (6, 20). Hysterectomy is a reasonable option for patients who do not wish to preserve their fertility, but it does not prevent metastasis (20). Furthermore, in patients with extensive uterine tumor, hysterectomy may substantially reduce the trophoblastic tumor burden (20) and thereby limit the need for multiple courses of chemotherapy . In our case, due to unstable and uterine rupture, hysterectomy was done but the need for chemotherapy courses was not removed and after several courses of chemotherapy with ema - co courses, it was cured . However, final diagnosis of our patient was invasive mole with metastasis to ovary and omentum and lung (stage 3) and uterine rupture . The patient responded to the treatment well and in follow up this patient was still fine for 5 years after definite care for metastasis invasive mole with surgery and chemotherapy . An invasive mole in uterus with ovarian and omental metastasis was diagnosed and the patient was successfully treated by hysterectomy and chemotherapy and she was fine for 5 years in follow up.

An expanded research design and methods section and supplementary figures can be found in an online data supplement available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1293/dc1 . Details on glucose and insulin tolerance testing, plasma insulin level determination, lipid metabolite measurement, metabolomics, exercise capacity studies, whole - body in vivo metabolic assessment, and immunoblot analysis are provided in the online data supplement . All animals received care according to the canadian council on animal care and the university of alberta health sciences animal welfare committee . Twelve - week - old c57bl/6 mice were placed on a standard chow / low - fat diet (4% kcal from lard) or high - fat diet (60% kcal from lard, research diets; d12492) for a 12-week period . At the end of week 12, animals were injected intraperitoneally every other day with the spt1 inhibitors, myriocin (0.5 mg / kg) suspended in 1x pbs, l - cycloserine (25 mg / kg) suspended in 1x pbs, or vehicle control for a 4-week period . At the end of the 4-week treatment protocol, animals were killed via intraperitoneal injection of sodium pentobarbital (12 mg) in the fed state in the middle of the dark cycle . Tissues were excised and immediately frozen in liquid n2 . In another study, 6-week - old db / db mice and their heterozygous controls (db/+) (jackson laboratories) as expected, mice fed a high - fat diet for 12 weeks became obese as indicated by a significant increase in weight gain, (supplementary fig . D). Diet - induced insulin - resistant and lean mice were placed in a comprehensive lab animal - monitoring system (clams) for whole - body metabolic assessment, which demonstrated a high - fat diet induced shift in fuel preference toward fatty acids as an oxidative energy source, indicated by the large drop in the respiratory exchange ratio (rer) (supplementary fig . Further support for an increase in fatty acid oxidation in obese mice is seen with the increase in gastrocnemius -hydroxyacyl - coa dehydrogenase (had) activity (table 1). Contrary to previous findings (16,17), we also report here that obesity induced by chronic high - fat feeding impairs whole - body oxygen consumption rates (supplementary fig . 2c and d). -hydroxyacyl - coa dehydrogenase (had) activity in gastrocnemius muscle of lean and obese mice treated with vehicle control or myriocin values reported are mol / g wet weight / min of n = 5 mice . Differences were determined using a two - way anova followed by bonferroni post hoc analysis . * significantly different from the low - fat counterpart . After 12 weeks of low- or high - fat diet, mice were treated with either myriocin (0.5 mg / kg every other day) or vehicle control . After 2 weeks of treatment, we demonstrated that inhibition of spt1 with myriocin reverses diet - induced insulin resistance, as determined by glucose tolerance and insulin tolerance testing (fig . If these protective effects took place at the skeletal muscle level, a group of animals were killed at 30 min after insulin injection during the insulin tolerance test, and muscles were excised and harvested for immunoblot analysis of the insulin - signaling pathway . We demonstrate that insulin stimulation of both akt and glycogen synthase kinase 3 (gsk3) phosphorylation were significantly improved in the gastrocnemius muscle of obese mice treated with myriocin (fig . Phosphorylation of 5amp activated protein kinase (ampk), another key signaling molecule regulating glucose metabolism, did not differ in gastrocnemius muscle of control and myriocin - treated obese mice (data not shown). Induced insulin resistance and improves insulin signaling . A: glucose tolerance test in low - fat fed and obese insulin - resistant mice treated with either vehicle control or myriocin . C: insulin tolerance test in low - fat diet and obese insulin - resistant mice treated with either vehicle control or myriocin . E: insulin - stimulated akt phosphorylation at serine 473, and (f) gsk3 phosphorylation at serine 9 in gastrocnemius muscle of obese insulin - resistant mice treated with either vehicle control or myriocin . Values represent mean se (n = 812 for a d; n = 4 for e and f). Differences were determined using either a two - tailed student t test or a two - way anova followed by a bonferroni post hoc analysis . * p <0.05, significantly different from all other groups . Myriocin treatment was without effect on food intake, body weight, and plasma insulin levels, but did reduce both postprandial and fasted plasma glucose levels in obese, insulin - resistant mice (table 2). Although fasting plasma insulin levels did not differ between diet - induced obesity (dio) mice treated with vehicle - control or myriocin, more sophisticated studies monitoring the changes in plasma insulin in response to a meal tolerance test in the obese jcr: la cp rat illustrate a significant improvement in plasma insulin control after treatment with the spt1 inhibitor, l - cycloserine (supplementary fig . 3). Interestingly, indirect calorimetry revealed that the improved insulin sensitivity in dio mice treated with myriocin was not associated with a decrease in fatty acid oxidation and an increase in carbohydrate oxidation, as similar rer values were observed between the dio control and myriocin - treated animals (fig . 2). Effect of myriocin on body and tissue weight, and plasma glucose and insulin levels in lean and obese mice values reported are from n = 511 mice . Differences were determined using a two - way anova followed by bonferroni post hoc analysis . * significantly different from the low - fat counterpart; significantly different from high - fat control . Twenty - four - hour (a), dark cycle (b), and light cycle respiratory exchange ratio (c) in low - fat fed and obese insulin - resistant mice treated with either vehicle control or myriocin . Differences were determined using a two - way anova followed by a bonferroni post hoc analysis . * p <0.05, significantly different from the low - fat diet counterpart . In a parallel series of experiments, after 12 weeks of high - fat feeding, mice were treated with either the spt1 inhibitor, l - cycloserine (25 mg / kg every other day) or vehicle control . Although not as dramatic as the results observed with myriocin, at 2 weeks after treatment, we report improvements in glucose and insulin tolerance in mice treated with l - cycloserine (supplementary fig . Obese, insulin - resistant mice were run on an exercise treadmill to determine exercise capacity . As expected, obese mice showed a dramatic reduction in both their treadmill time and distance when compared with their lean counterparts (fig . Interestingly, treatment of obese mice for 2 weeks with myriocin reversed this reduction in exercise capacity (fig . 3a and b). This improvement in exercise capacity observed in obese mice treated with myriocin can be explained by enhanced whole - body oxygen consumption rates compared with their control counterparts (fig . C). In addition, we observed greater citrate synthase activity in gastrocnemius muscle of obese mice treated with myriocin (fig . Protein expression of peroxisome proliferator - activated receptor- coactivator-1 (pgc1), a transcriptional coactivator that plays a key role in regulating a number of genes involved in energy metabolism (18), showed a trend toward a reduction in control - treated dio mice (p = 0.077) that was not apparent in myriocin - treated dio mice (fig . Furthermore, we also demonstrate that pretreatment with myriocin increases citrate synthase activity in c2c12 myotubes exposed to 1.0 mmol / l palmitate for 16 h (fig . These observations illustrate improvements in mitochondrial function, possibly explaining why exercise capacity and whole - body oxygen consumption rates were enhanced in this group . Time (a) and distance (b) during an exercise capacity challenge on a running treadmill . Differences were determined using a two - way anova followed by a bonferroni post hoc analysis . * p <0.05, significantly different from the low - fat diet counterpart . Myriocin treatment reverses the impairment in whole - body oxygen consumption rates caused by dio . C: twenty - four hour (a), dark cycle (b), and light cycle (c) whole - body oxygen consumption assessment in low - fat diet and obese insulin - resistant mice treated with either vehicle control or myriocin . D: gastrocnemius muscle citrate synthase activity in vehicle control and myriocin - treated dio mice . E: pgc1 expression in low - fat diet and obese insulin - resistant mice treated with either vehicle control or myriocin . F: citrate synthase activity in vehicle control and myriocin - pretreated c2c12 skeletal muscle myotubes exposed to 1.0 mmol / l palmitate for 16 h. values represent mean se (n = 512). Differences were determined using either a two - tailed student t test or a two - way anova followed by a bonferroni post hoc analysis . * p <0.05, significantly different from the high - fat diet control mice . Auc, area under the curve . Metabolic profiling of mice provided further insight with regards to mitochondrial function in obese, insulin - resistant mice, as control - treated dio mice had a significant increase in long - chain acyl carnitine esters versus their lean counterparts (table 3), indicative of mitochondrial overload and the incomplete oxidation of fatty acids (19). However, the accumulation of long - chain acyl carnitine esters in myriocin - treated dio mice was even greater (table 3). This suggests that incomplete fatty acid oxidation rates were even more pronounced in the myriocin - treated dio mice, but these animals also had a significant reduction in short - chain acyl carnitine ester content (table 3), which is consistent with long - chain acyl - coa dehydrogenase inhibition and reduced oxidation of long - chain fatty acids . Metabolic profiling of gastrocnemius muscle from lean and obese mice treated with either vehicle control or myriocin values reported are in pmol / mg protein from n = 6 mice . Differences were determined using a two - way anova followed by bonferroni post hoc analysis . * significantly different from the lf counterpart . After 3 weeks of treatment with myriocin, in vivo heat production and ambulatory activity were assessed in our clams apparatus . Paralleling our observations with regard to whole - body oxygen consumption rates, obesity caused a decline in whole - body heat production that was reversed by myriocin treatment (supplementary fig . Moreover, obesity - induced insulin resistance was associated with reductions in physical activity that were not altered by myriocin treatment (supplementary fig . Investigation of the lipid metabolite profile in gastrocnemius muscle demonstrated that chronic high - fat feeding increased long - chain acyl - coa, ceramide and dag content, but only a trend to an increase in tag content was observed (fig . D). Treatment with myriocin in obese mice increased gastrocnemius tag content in comparison to their low - fat counterparts, but did not change the dio - associated rise in long - chain acyl - coa and dag content, and as expected, resulted in a dramatic reduction in ceramide content (fig . D). These results suggest a key role for ceramide in mediating skeletal muscle insulin resistance, and indicate that the other lipid metabolites possibly may not be as important in the insulin - resistance development . Further support for this statement is seen with the positive correlation between ceramide content and the area under the curve during the glucose tolerance test, whereas no correlation was observed with any of the other lipid metabolites (fig . Interestingly, in a previous study, we showed that mice deficient for malonyl coa decarboxylase (mcd/) are protected from obesity - induced glucose intolerance and insulin resistance, which was associated with a reduction in incomplete fatty acid oxidation rates (19). In this study we show that these same mcd/ mice do not accumulate ceramide in their gastrocnemius muscle after 12 weeks of high - fat feeding (fig . 6), although they did accumulate other lipid metabolites such as long - chain acyl - coa (19). Inhibition of spt1 reduces skeletal muscle ceramide levels with no effect on other lipid metabolites . D: gastrocnemius triacylglycerol (tag) (a), long - chain acyl - coa (b), ceramide (c), and diacylglycerol (d) levels in low - fat fed and obese insulin - resistant mice treated with either vehicle control or myriocin . Differences were determined using a two - way anova followed by bonferroni post hoc analysis . * p <0.05, significantly different from the low - fat diet counterpart . P <0.05, significantly different from the high - fat diet control mice . H: correlation between the respective areas under the curve during the glucose tolerance test and ceramide (e), tag (f), long - chain acyl - coa (g), and diacylglycerol (h) content of (n = 1418) samples . Malonyl coa decarboxylase - deficient mice (mcd/) do not accumulate skeletal muscle ceramide after 12 weeks of high - fat feeding . A: area under the curve during a glucose tolerance test after 12 weeks of high - fat feeding in wild - type and mcd/ mice . B: corresponding gastrocnemius ceramide levels in mcd/ mice after 12 weeks of high - fat feeding . Differences were determined using a two - way anova followed by bonferroni post hoc analysis . P <0.05, significantly different from the high - fat diet wild - type mice . Auc, area under the curve . To determine if ceramides may also be involved in genetic forms of insulin resistance and type 2 diabetes, we treated leptin receptor deficient (db / db) mice with myriocin to see if we could prevent the progression of insulin resistance in these animals . We split db / db mice at 6 weeks of age into two groups, and ensured that there were no differences in glucose tolerance before initiating treatment with myriocin (fig . Both the db / db control and myriocin - treated groups experienced similar body weight increases after 2 weeks of treatment (data not shown), however, although the db / db control group became glucose intolerant, the db / db group treated with myriocin did not (fig . Fasting blood glucose levels were also significantly lower in the db / db mice treated with myriocin, and although their response to insulin was delayed, myriocin - treated db / db mice demonstrated lower blood glucose levels at nearly all time points during an insulin tolerance test (fig . 7d f). Placing these animals in the clams apparatus yielded a profile similar to that of the dio mice . The db / db controls had a lower rer in the dark cycle than db/+ lean mice, and had lower whole - body oxygen consumption rates and ambulatory activity, but no change in overall heat production; interestingly, myriocin treatment of db / db mice did not restore any of these altered parameters in db / db controls, except for a restoration of whole - body oxygen consumption rates during the light cycle (fig . Examination of the lipid metabolite profile revealed that tag and long - chain acyl - coa levels were elevated in gastrocnemius muscle of db / db controls versus db/+ lean mice, whereas, unexpectedly, dag and ceramide levels were similar between the two groups (fig . H). Myriocin treatment of db / db mice had no effect on tag, long - chain acyl - coa, or dag levels in gastrocnemius muscle versus db / db control mice, but did lead to a dramatic reduction in ceramide levels (fig . Insulin - stimulated akt and gsk3 phosphorylation were also depressed in db / db control versus db/+ lean mice, but showed an improvement in db / db mice treated with myriocin (fig . Prevention of insulin resistance in db / db mice via myriocin treatment . A: pretreatment glucose tolerance test (gtt) in db / db mice at 6 weeks of age . C: respective areas under the curve for the post - treatment gtt in db / db mice . D: insulin tolerance test (itt) in db / db mice treated with vehicle control or myriocin . F: fed and fasted plasma glucose levels in db / db mice treated with vehicle control or myriocin . Differences were determined using either a two - tailed student t test, or a one - way or two - way anova followed by bonferroni post hoc analysis . * p <0.05, significantly different from the db / db control mice . In vivo metabolic parameters, intramyocellular lipid metabolite profile, and insulin signaling in db / db mice treated with myriocin . Rer (a), whole - body oxygen consumption (b), heat production (c), and ambulatory activity (d) in db/+ heterozygous mice, and db / db mice treated with vehicle control or myriocin . Gastrocnemius triacylglycerol (e), long - chain acyl - coa (f), diacylglycerol (g), and ceramide levels (h) in db/+ heterozygous mice, and db / db mice treated with vehicle control or myriocin . I: insulin stimulated akt phosphorylation at serine 473, and (j) gsk3 phosphorylation at serine 9 in gastrocnemius muscle of db/+ heterozygous mice and db / db mice treated with vehicle control or myriocin . Differences were determined using either a one - way or two - way anova followed by bonferroni post hoc analysis . * p <0.05, significantly different from the db / db control mice . As expected, mice fed a high - fat diet for 12 weeks became obese as indicated by a significant increase in weight gain, (supplementary fig . D). Diet - induced insulin - resistant and lean mice were placed in a comprehensive lab animal - monitoring system (clams) for whole - body metabolic assessment, which demonstrated a high - fat diet induced shift in fuel preference toward fatty acids as an oxidative energy source, indicated by the large drop in the respiratory exchange ratio (rer) (supplementary fig . Further support for an increase in fatty acid oxidation in obese mice is seen with the increase in gastrocnemius -hydroxyacyl - coa dehydrogenase (had) activity (table 1). Contrary to previous findings (16,17), we also report here that obesity induced by chronic high - fat feeding impairs whole - body oxygen consumption rates (supplementary fig . 2c and d). -hydroxyacyl - coa dehydrogenase (had) activity in gastrocnemius muscle of lean and obese mice treated with vehicle control or myriocin values reported are mol / g wet weight / min of n = 5 mice . Differences were determined using a two - way anova followed by bonferroni post hoc analysis . * significantly different from the low - fat counterpart . After 12 weeks of low- or high - fat diet, mice were treated with either myriocin (0.5 mg / kg every other day) or vehicle control . After 2 weeks of treatment, we demonstrated that inhibition of spt1 with myriocin reverses diet - induced insulin resistance, as determined by glucose tolerance and insulin tolerance testing (fig . If these protective effects took place at the skeletal muscle level, a group of animals were killed at 30 min after insulin injection during the insulin tolerance test, and muscles were excised and harvested for immunoblot analysis of the insulin - signaling pathway . We demonstrate that insulin stimulation of both akt and glycogen synthase kinase 3 (gsk3) phosphorylation were significantly improved in the gastrocnemius muscle of obese mice treated with myriocin (fig . Phosphorylation of 5amp activated protein kinase (ampk), another key signaling molecule regulating glucose metabolism, did not differ in gastrocnemius muscle of control and myriocin - treated obese mice (data not shown). Inhibition of serine palmitoyl transferase 1 (spt1) reverses high - fat diet induced insulin resistance and improves insulin signaling . A: glucose tolerance test in low - fat fed and obese insulin - resistant mice treated with either vehicle control or myriocin . C: insulin tolerance test in low - fat diet and obese insulin - resistant mice treated with either vehicle control or myriocin . E: insulin - stimulated akt phosphorylation at serine 473, and (f) gsk3 phosphorylation at serine 9 in gastrocnemius muscle of obese insulin - resistant mice treated with either vehicle control or myriocin . Values represent mean se (n = 812 for a d; n = 4 for e and f). Differences were determined using either a two - tailed student t test or a two - way anova followed by a bonferroni post hoc analysis . * p <0.05, significantly different from all other groups . Myriocin treatment was without effect on food intake, body weight, and plasma insulin levels, but did reduce both postprandial and fasted plasma glucose levels in obese, insulin - resistant mice (table 2). Although fasting plasma insulin levels did not differ between diet - induced obesity (dio) mice treated with vehicle - control or myriocin, more sophisticated studies monitoring the changes in plasma insulin in response to a meal tolerance test in the obese jcr: la cp rat illustrate a significant improvement in plasma insulin control after treatment with the spt1 inhibitor, l - cycloserine (supplementary fig . 3). Interestingly, indirect calorimetry revealed that the improved insulin sensitivity in dio mice treated with myriocin was not associated with a decrease in fatty acid oxidation and an increase in carbohydrate oxidation, as similar rer values were observed between the dio control and myriocin - treated animals (fig . 2). Effect of myriocin on body and tissue weight, and plasma glucose and insulin levels in lean and obese mice values reported are from n = 511 mice . Differences were determined using a two - way anova followed by bonferroni post hoc analysis . * significantly different from the low - fat counterpart; significantly different from high - fat control . Twenty - four - hour (a), dark cycle (b), and light cycle respiratory exchange ratio (c) in low - fat fed and obese insulin - resistant mice treated with either vehicle control or myriocin . Differences were determined using a two - way anova followed by a bonferroni post hoc analysis . * p <0.05, significantly different from the low - fat diet counterpart . In a parallel series of experiments, after 12 weeks of high - fat feeding, mice were treated with either the spt1 inhibitor, l - cycloserine (25 mg / kg every other day) or vehicle control . Although not as dramatic as the results observed with myriocin, at 2 weeks after treatment, we report improvements in glucose and insulin tolerance in mice treated with l - cycloserine (supplementary fig . Obese, insulin - resistant mice were run on an exercise treadmill to determine exercise capacity . As expected, obese mice showed a dramatic reduction in both their treadmill time and distance when compared with their lean counterparts (fig . Interestingly, treatment of obese mice for 2 weeks with myriocin reversed this reduction in exercise capacity (fig . 3a and b). This improvement in exercise capacity observed in obese mice treated with myriocin can be explained by enhanced whole - body oxygen consumption rates compared with their control counterparts (fig . C). In addition, we observed greater citrate synthase activity in gastrocnemius muscle of obese mice treated with myriocin (fig . Protein expression of peroxisome proliferator - activated receptor- coactivator-1 (pgc1), a transcriptional coactivator that plays a key role in regulating a number of genes involved in energy metabolism (18), showed a trend toward a reduction in control - treated dio mice (p = 0.077) that was not apparent in myriocin - treated dio mice (fig . Furthermore, we also demonstrate that pretreatment with myriocin increases citrate synthase activity in c2c12 myotubes exposed to 1.0 mmol / l palmitate for 16 h (fig . These observations illustrate improvements in mitochondrial function, possibly explaining why exercise capacity and whole - body oxygen consumption rates were enhanced in this group . Time (a) and distance (b) during an exercise capacity challenge on a running treadmill . Differences were determined using a two - way anova followed by a bonferroni post hoc analysis . * p <0.05, significantly different from the low - fat diet counterpart . Myriocin treatment reverses the impairment in whole - body oxygen consumption rates caused by dio . C: twenty - four hour (a), dark cycle (b), and light cycle (c) whole - body oxygen consumption assessment in low - fat diet and obese insulin - resistant mice treated with either vehicle control or myriocin . D: gastrocnemius muscle citrate synthase activity in vehicle control and myriocin - treated dio mice . E: pgc1 expression in low - fat diet and obese insulin - resistant mice treated with either vehicle control or myriocin . F: citrate synthase activity in vehicle control and myriocin - pretreated c2c12 skeletal muscle myotubes exposed to 1.0 mmol / l palmitate for 16 h. values represent mean se (n = 512). Differences were determined using either a two - tailed student t test or a two - way anova followed by a bonferroni post hoc analysis . * p <0.05, significantly different from the low - fat diet counterpart . P <0.05, significantly different from the high - fat diet control mice . Auc, area under the curve . Metabolic profiling of mice provided further insight with regards to mitochondrial function in obese, insulin - resistant mice, as control - treated dio mice had a significant increase in long - chain acyl carnitine esters versus their lean counterparts (table 3), indicative of mitochondrial overload and the incomplete oxidation of fatty acids (19). However, the accumulation of long - chain acyl carnitine esters in myriocin - treated dio mice was even greater (table 3). This suggests that incomplete fatty acid oxidation rates were even more pronounced in the myriocin - treated dio mice, but these animals also had a significant reduction in short - chain acyl carnitine ester content (table 3), which is consistent with long - chain acyl - coa dehydrogenase inhibition and reduced oxidation of long - chain fatty acids . Metabolic profiling of gastrocnemius muscle from lean and obese mice treated with either vehicle control or myriocin values reported are in pmol / mg protein from n = 6 mice . Differences were determined using a two - way anova followed by bonferroni post hoc analysis . * significantly different from the lf counterpart . After 3 weeks of treatment with myriocin, in vivo heat production and ambulatory activity were assessed in our clams apparatus . Paralleling our observations with regard to whole - body oxygen consumption rates, obesity caused a decline in whole - body heat production that was reversed by myriocin treatment (supplementary fig . Moreover, obesity - induced insulin resistance was associated with reductions in physical activity that were not altered by myriocin treatment (supplementary fig . Investigation of the lipid metabolite profile in gastrocnemius muscle demonstrated that chronic high - fat feeding increased long - chain acyl - coa, ceramide and dag content, but only a trend to an increase in tag content was observed (fig . D). Treatment with myriocin in obese mice increased gastrocnemius tag content in comparison to their low - fat counterparts, but did not change the dio - associated rise in long - chain acyl - coa and dag content, and as expected, resulted in a dramatic reduction in ceramide content (fig . D). These results suggest a key role for ceramide in mediating skeletal muscle insulin resistance, and indicate that the other lipid metabolites possibly may not be as important in the insulin - resistance development . Further support for this statement is seen with the positive correlation between ceramide content and the area under the curve during the glucose tolerance test, whereas no correlation was observed with any of the other lipid metabolites (fig . H). Interestingly, in a previous study, we showed that mice deficient for malonyl coa decarboxylase (mcd/) are protected from obesity - induced glucose intolerance and insulin resistance, which was associated with a reduction in incomplete fatty acid oxidation rates (19). In this study we show that these same mcd/ mice do not accumulate ceramide in their gastrocnemius muscle after 12 weeks of high - fat feeding (fig . 6), although they did accumulate other lipid metabolites such as long - chain acyl - coa (19). Inhibition of spt1 reduces skeletal muscle ceramide levels with no effect on other lipid metabolites . D: gastrocnemius triacylglycerol (tag) (a), long - chain acyl - coa (b), ceramide (c), and diacylglycerol (d) levels in low - fat fed and obese insulin - resistant mice treated with either vehicle control or myriocin . Differences were determined using a two - way anova followed by bonferroni post hoc analysis . * p <0.05, significantly different from the low - fat diet counterpart . P <0.05, significantly different from the high - fat diet control mice . H: correlation between the respective areas under the curve during the glucose tolerance test and ceramide (e), tag (f), long - chain acyl - coa (g), and diacylglycerol (h) content of (n = 1418) samples . Malonyl coa decarboxylase - deficient mice (mcd/) do not accumulate skeletal muscle ceramide after 12 weeks of high - fat feeding . A: area under the curve during a glucose tolerance test after 12 weeks of high - fat feeding in wild - type and mcd/ mice . B: corresponding gastrocnemius ceramide levels in mcd/ mice after 12 weeks of high - fat feeding . Differences were determined using a two - way anova followed by bonferroni post hoc analysis . * p <0.05, significantly different from low - fat diet counterpart . P <0.05, significantly different from the high - fat diet wild - type mice . To determine if ceramides may also be involved in genetic forms of insulin resistance and type 2 diabetes, we treated leptin receptor deficient (db / db) mice with myriocin to see if we could prevent the progression of insulin resistance in these animals . We split db / db mice at 6 weeks of age into two groups, and ensured that there were no differences in glucose tolerance before initiating treatment with myriocin (fig . Both the db / db control and myriocin - treated groups experienced similar body weight increases after 2 weeks of treatment (data not shown), however, although the db / db control group became glucose intolerant, the db / db group treated with myriocin did not (fig . Fasting blood glucose levels were also significantly lower in the db / db mice treated with myriocin, and although their response to insulin was delayed, myriocin - treated db / db mice demonstrated lower blood glucose levels at nearly all time points during an insulin tolerance test (fig . F). Placing these animals in the clams apparatus yielded a profile similar to that of the dio mice . The db / db controls had a lower rer in the dark cycle than db/+ lean mice, and had lower whole - body oxygen consumption rates and ambulatory activity, but no change in overall heat production; interestingly, myriocin treatment of db / db mice did not restore any of these altered parameters in db / db controls, except for a restoration of whole - body oxygen consumption rates during the light cycle (fig . Examination of the lipid metabolite profile revealed that tag and long - chain acyl - coa levels were elevated in gastrocnemius muscle of db / db controls versus db/+ lean mice, whereas, unexpectedly, dag and ceramide levels were similar between the two groups (fig . H). Myriocin treatment of db / db mice had no effect on tag, long - chain acyl - coa, or dag levels in gastrocnemius muscle versus db / db control mice, but did lead to a dramatic reduction in ceramide levels (fig . Insulin - stimulated akt and gsk3 phosphorylation were also depressed in db / db control versus db/+ lean mice, but showed an improvement in db / db mice treated with myriocin (fig . Prevention of insulin resistance in db / db mice via myriocin treatment . A: pretreatment glucose tolerance test (gtt) in db / db mice at 6 weeks of age . C: respective areas under the curve for the post - treatment gtt in db / db mice . D: insulin tolerance test (itt) in db / db mice treated with vehicle control or myriocin . F: fed and fasted plasma glucose levels in db / db mice treated with vehicle control or myriocin . Differences were determined using either a two - tailed student t test, or a one - way or two - way anova followed by bonferroni post hoc analysis . * p <0.05, significantly different from the db / db control mice . In vivo metabolic parameters, intramyocellular lipid metabolite profile, and insulin signaling in db / db mice treated with myriocin . Rer (a), whole - body oxygen consumption (b), heat production (c), and ambulatory activity (d) in db/+ heterozygous mice, and db / db mice treated with vehicle control or myriocin . Gastrocnemius triacylglycerol (e), long - chain acyl - coa (f), diacylglycerol (g), and ceramide levels (h) in db/+ heterozygous mice, and db / db mice treated with vehicle control or myriocin . I: insulin stimulated akt phosphorylation at serine 473, and (j) gsk3 phosphorylation at serine 9 in gastrocnemius muscle of db/+ heterozygous mice and db / db mice treated with vehicle control or myriocin . Differences were determined using either a one - way or two - way anova followed by bonferroni post hoc analysis . * p <0.05, significantly different from the db / db control mice . Our results show that inhibition of spt1 reduces de novo ceramide synthesis in muscle, which has novel effects on whole - body energy metabolism and is associated with a profound reversal of glucose intolerance and insulin resistance induced by chronic high - fat feeding . Furthermore, we show that these improvements are dissociated from the other lipid metabolites believed to play a role in the development of insulin resistance . Interestingly, obesity - induced insulin resistance in mice is associated with a detriment in aerobic exercise capacity and whole - body oxygen consumption rates, both of which are partially reversed via spt1 inhibition . Previous studies have postulated that skeletal muscle insulin resistance is caused by the intramyocellular cytosolic accumulation of lipid metabolites (tag, long - chain acyl - coa, dag, ceramide, etc .) In particular, long - chain acyl - coa and dag have received considerable attention because of their ability to activate the classic / novel protein kinase c signaling cascade, which can phosphorylate insulin receptor substrate proteins on serine residues, preventing their activation via the insulin receptor (4,5,8,2123). It is important to note, however, that most (95%) acyl - coa esters are located inside the mitochondria (24,25), suggesting that if long - chain acyl - coa accumulation does play a role toward insulin resistance development, it is possible that mitochondrial, as opposed to cytosolic long - chain acyl - coa, is the primary contributor . Although tag has been shown in numerous studies to be elevated in muscle in association with the development of insulin resistance, recent studies have shown that tag may actually serve as a buffer, protecting the muscle against the accumulation of the more reactive lipid metabolite species (10,11). In regards to ceramide, data are mixed with its role in insulin resistance development, because in some studies, ceramide accumulation is not evident in muscle (5,26), and in other studies where accumulation does occur, the relative increase in the ceramide pool is not that large (12,27). (12) has shed some light on this issue, as they demonstrated that ceramide accumulation in muscle is dependent on the type of diet fed to the animals . In particular, saturated fatty acids drive de novo ceramide synthesis in muscle via spt1, whereas unsaturated fatty acids cause insulin resistance via other mechanisms (12). Such findings may potentially explain why ceramide accumulation is not observed in studies of insulin resistance where the model employed is a lipid infusion that consists primarily of unsaturated fatty acids (22). Furthermore, holland et al . (12) showed in their study that preventing de novo synthesis of ceramide via spt1 inhibition with myriocin prevented the development of glucose intolerance in obese zucker rats, and prevented the palmitate - induced inhibition of insulin - stimulated 2-deoxyglucose uptake in isolated soleus muscle . (28) also reported positive findings with myriocin treatment in leptin - deficient and dio mice, providing further support that ceramide plays a key role in the development of insulin resistance . Interestingly, these authors also observed a weight loss effect due to myriocin treatment that we did not observe in our studies . However, the authors in this study used a much longer treatment than ours (8 vs. 4 weeks), and noted that they did not observe a weight loss effect until later in the treatment period . Furthermore, 3 weeks of myriocin treatment in dio mice improved hyperglycemia and whole - body oxygen consumption rates in their mice, despite no change in body weight compared with control - treated dio mice, which is consistent with our results in dio mice treated with myriocin for 2 weeks . Yang et al . Also observed a dramatic reduction in hepatic steatosis that is consistent with our observations in regards to hepatic tag content . Our study adds further support to the studies examining the role of ceramide in mediating insulin resistance (12,28) by illustrating the potential for targeting spt1 as a treatment against insulin resistance . . Moreover, by examining other lipid metabolites such as tag, dag, long - chain acyl - coa, and acyl carnitine content in skeletal muscle, we are able to discern important differences with regard to the relative importance of each metabolite toward the development of skeletal mucle insulin resistance . Importantly, reductions in skeletal muscle ceramide accumulation may represent a potential explanation for the exercise paradox observed in humans . Dube et al . (15) showed that obese, insulin - resistant men placed on an aerobic exercise training regime have elevated intramyocellular lipid and tag stores . However, marked reductions in muscle ceramide levels are observed, which may explain the enhanced insulin sensitivity of these men . Moreover, bruce et al . (14) showed that the improved insulin sensitivity observed with exercise training in humans is associated with a drop in muscle ceramide levels, and in particular, the saturated species . (29) showed that exercise training of rats leads to a dramatic drop in the saturated species of ceramide in muscle, which is associated with an enhanced 2-deoxyglucose uptake . In addition, mice overexpressing diacylglycerol acyl transferase in muscle are protected from high - fat - diet induced insulin resistance and palmitate inhibition of 2-deoxyglucose uptake in isolated muscle, both of which are associated with an elevation of muscle tag and drop in ceramide levels (10). Our results support these studies, as we show that obese, insulin - resistant mice treated with myriocin had significant increases in intramyocellular tag, long - chain acyl - coa, and dag, but a dramatic drop in ceramide content . Moreover, we observed a positive correlation with ceramide content and glucose intolerance, but not with any of the other lipid metabolites . We believe that with this finding, in the setting of obesity, that ceramide may be more vital to the development of skeletal muscle insulin resistance than the other lipid metabolites . Support for this statement is also evident in culture models of ceramide accumulation, whereby inhibition of spt1 was able to prevent palmitate - induced insulin resistance in both human and rat l6 myotubes, despite elevated tag and dag levels (11,13). Furthermore, a recent study in humans demonstrated that insulin resistant muscle is associated with elevated ceramide content, but no change in dag content (30). Nonetheless, it is also important to note that our measurement of dag assessed total cellular levels of dag, and it is possible that differences in plasma membrane dag were significantly reduced via myriocin treatment . Because dag at the membrane is believed to be the specific dag pool responsible for mediating skeletal muscle insulin resistance (3), it will be important for future studies to investigate this in more detail . One of the most surprising findings of this study was that chronic high - fat feeding resulted in a dramatic decline in whole - body oxygen consumption rates . The majority of studies that have examined the effect of high - fat feeding on whole - body oxygen consumption rates via use of the clams apparatus have reported elevations in oxygen consumption rates (16,17). Although the differences between these studies and ours could be due to the duration or composition of the diet, we propose two possible explanations for this observation of ours . First, it has been reported that obesity - induced insulin resistance causes mitochondrial dysfunction that results from an impairment of fatty acid oxidative capacity (58). Although it may be possible that our model of insulin resistance is inducing mitochondrial dysfunction, it is highly unlikely due to impairments in muscle fatty acid oxidative capacity, as the rer values in obese mice reported in this study are very close to 0.7, indicating that these animals have no trouble utilizing fat as an energy source . Nonetheless, other factors, such as mitochondrial content, protein expression of electron transport chain (etc) complexes, or activity of these complexes, may account for potential mitochondrial dysfunction and the subsequent impairment of oxygen consumption rates observed in obese mice (31,32). However, we did not observe differences in protein expression of cytochrome c of the etc in any group (data not shown). Second, and just as relevant to the findings of this study, is that obesity - induced insulin resistance has been associated with elevated rates of incomplete fatty acid oxidation, which can arise when rates of fatty acid oxidation are disconnected from tca cycle activity (19,33,34). This disconnect arises due to the sedentary nature of obese individuals and animals, thus there is no demand for the tca cycle to upregulate its activity to deal with the increased fatty acid supply that is being utilized as an energy source (19,33,34). If the tca cycle is unable to accommodate the increasing acetyl coa coming from fatty acid oxidation, reducing equivalents such as nadh and fadh2 would not donate their electrons to the complexes of the etc, accounting for the reduction in oxygen consumption rates . Our observation of increased accumulation of long - chain acyl carnitine esters in the muscle of dio mice is thus consistent with elevated rates of incomplete fatty acid oxidation . In contrast, there was an even greater accumulation of long - chain acyl carnitine esters in myriocin - treated dio mice, which at first glance would suggest even greater rates of incomplete fatty acid oxidation in these animals . However, myriocin - treated dio mice actually had a significant reduction in the content of a number of short - chain acyl carnitine esters, and this, in combination with the rise of long - chain acyl carnitine esters, is suggestive of long - chain acyl - coa dehydrogenase and subsequent long - chain fatty acid oxidation inhibition (35). Another piece of indirect support for fatty acid oxidation inhibition with myriocin treatment in dio mice is the observation that tag accumulated in the muscle of these animals versus their lean counterparts, but not in control - treated dio mice versus their lean counterparts . A reduction in fatty acid oxidation - derived nadh would decrease nadh / nadph oxidase activity and subsequent superoxide production in myriocin - treated dio mice, which would contribute toward their improved mitochondrial function . This improvement in mitochondrial function, coupled together with improvements in glucose metabolism and glucose - derived acetyl coa production for the tca cycle, may contribute to the greater oxygen consumption rates in these animals . Obesity - induced decrements in pgc1 protein expression might also explain impairments in mitochondrial function (34,36,37), and although not significant, we observed a trend toward a reduction in gastrocnemius pgc1 protein expression in control - treated dio mice (p = 0.077) that was not evident in myriocin - treated dio mice . Interestingly, citrulline levels were increased in myriocin - treated dio mice versus their control counterparts (supplementary fig . A previous study in humans showed that supplementation of citrulline enhances aerobic oxidative metabolism (38), supporting our findings of increased whole - body oxygen consumption rates and greater exercise time in myriocin - treated dio mice . How myriocin and subsequent spt1 inhibition would influence skeletal muscle citrulline levels is currently unknown, but is undoubtedly an intriguing avenue for future investigation . In addition, we have previously shown that mcd/ mice (a genetic model of fatty acid oxidation deficiency) are protected from obesity - induced insulin resistance . Interestingly, we show in this study that these exact same animals do not accumulate ceramide in their muscle after 12 weeks of high - fat feeding, leading to the very intriguing possibility that intramyocellular ceramide accumulation is linked to the mitochondrial dysfunction and enhanced skeletal muscle fatty acid oxidation rates observed in insulin resistance . A limitation with our interpretation of whole - body oxygen consumption rates is that, unlike human studies, we were unable to normalize our oxygen consumption rates to lean body mass . It is entirely possible that whole - body oxygen consumption rates were simply lower in dio mice because of a significant increase in overall adiposity, due to fat mass having a lower metabolic rate than lean body mass . However, the fact that adiposity and body weight were similar between myriocin- and control - treated dio mice suggests that this would not be a contributing factor to the higher oxygen consumption rates observed in the myriocin - treated dio mice . Although we believe that the changes accounting for the greater oxygen consumption rates in myriocin - treated dio mice primarily reflect the muscle, we cannot ignore possible contributions from changes in other peripheral tissues, such as brown adipose tissue and uncoupling protein activity . The beneficial effects mediated by inhibition of spt1 and prevention of de novo ceramide synthesis could also arise from liver effects in our animals . Regardless, we did not observe increases in hepatic ceramide content after diet - induced obesity, and myriocin treatment had no effect on insulin - stimulated akt and gsk3 phosphorylation in obese mice versus their respective controls (supplementary fig ., recent studies have also shown that high - fat feeding does not increase ceramide content in the liver (39), and increases in hepatic ceramide content via genetic overexpression of either dgat1 or dgat2 does not result in any type of insulin resistance or inflammation (40). Moreover, we reported no difference during a pyruvate challenge of fasted, obese, control- or myriocin - treated mice (supplementary fig . 8), suggesting that gluconeogenic capacity was not different between the two groups and that the liver likely does not play a key role with the improved insulin sensitivity observed in myriocin - treated mice . Regardless, we cannot entirely rule out the possibility that the liver plays a role with the benefit observed during myriocin treatment, as the dio - associated rise in hepatic tag content was reversed via myriocin treatment, and thus it will be important for future studies to delineate the role of hepatic spt1 in greater depth . Finally, chronic low - grade inflammation has been shown in a number of studies to play a role in causing obesity - induced insulin resistance (4143). Inflammatory and stress kinases, such as p38 mapk and jnk, have been proposed to be downstream mediators of this inflammatory effect, as inhibitors of both kinases are able to prevent high - fat diet induced insulin resistance (9,4446). Unexpectedly, the phosphorylation status of both p38 mapk and jnk was not altered by dio, nor was it altered by myriocin treatment (supplementary fig . 9), suggesting that inflammation may not play as vital a role in our model of insulin resistance . It may also be possible that inflammation in our model is mediated by some other kinase, such as ikk (47,48). With regard to the findings in db / db mice, we report very similar findings to what we observed in the obesity - induced insulin - resistant mice, and that treatment with myriocin also yielded a very similar beneficial profile . Interestingly, gastrocnemius ceramide levels, although reduced in myriocin - treated db / db mice, did not differ between db/+ lean and db / db control mice . This suggests, at least in this model, that perhaps ceramide metabolites, such as glucosylceramide, are more important in mediating skeletal muscle insulin resistance than ceramide itself (49). Furthermore, the ceramide pool is under a dynamic process of synthesis and degradation (9), and although de novo synthesis of ceramide may be increased in these animals, a simultaneous increase in ceramide degradation would mask out any noticeable change . In summary, we show that ceramide accumulation in skeletal muscle plays a key role during obesity - induced insulin resistance, whereas the other lipid metabolites, such as tag, long - chain acyl - coa, and dag, may not be as vital . Importantly, inhibition of de novo ceramide synthesis has novel effects on whole - body energy metabolism and is sufficient to reverse obesity - induced whole - body glucose intolerance and insulin resistance . Furthermore, whole - body oxygen consumption rates and exercise capacity in obese mice are improved via inhibition of de novo ceramide synthesis . Last, our finding that muscle ceramide levels are not elevated in db / db mice, but that inhibition of de novo ceramide synthesis still prevents their development of insulin resistance, suggests the possibility that ceramide metabolites may also play a role in the progression of this disease.

Antibodies against amp kinase (ampk), phosporylated ampk (pampk), acetyl coa carboxylase (acc), phosporylated acc, akt, phosporylated akt, and tubulin were from cell signaling; anti - oxphos was from mitosciences; anti cd36-[hrp] was purchased from novus biologicals, and human recombinant insulin (novolin) from novo nordisk canada . This study was performed with the approval of the university of alberta animal policy and welfare committee . Experiments were carried out on male wild - type (c57bl6) and cd36 knockout (ko) mice (19) maintained in a temperature - controlled room with a reversed 12-h light/12-h dark cycle . Mice were left relatively undisturbed for either 1214 or 5258 weeks of age with free access to water and standard rodent diet (category no . 5001; labdiet). At 3234 weeks of age, a subset of mice was randomly divided into a low - fat diet group (category no . D12450b; research diets) and a high - fat diet group (category no . D12492; research diets) for a period of 12 weeks . Indirect calorimetry was performed using the comprehensive lab animal monitoring system (oxymax / clams; columbus instruments, colombus, oh). Following an initial 24-h acclimatization period, mice were monitored every 13 min for 24 h to complete a 12-h dark (active)/12-h light (inactive) cycle . The respiratory exchange ratio (rer = vco2/vo2) was used to estimate the percent contribution of fat and carbohydrate to whole - body energy metabolism in mice in vivo . Total activity was calculated by adding z counts (rearing or jumping) to total counts associated with ambulatory movement and stereotypical behavior (grooming and scratching). Upon phospholipid digestion with phospholipase c (2 h at 30c) and lipid extraction, levels of triglycerides were determined in gastrocnemius muscle lysates by gas - liquid chromatography as previously described (20). Identification and quantification of the major long - chain acyl - coa molecular species (c16:0, c18:0, and c18:1) and c18-ceramides were performed by high - performance liquid chromatography as previously described (21). Overnight - fasted mice were anesthetized with sodium pentobarbital, and gastrocnemius muscle was rapidly removed and freeze - clamped in liquid nitrogen and stored at 80c . Acylcarnitine measurements were made using flow - injection tandem mass spectrometry as previously described (14), and organic acids were quantified as previously described (22). Comparisons between groups were performed using unpaired student's two - tailed t test or anova with a bonferroni post hoc test of pairwise comparisons where appropriate . A probability value of <0.05 is considered significant . For further descriptions of the materials and methods, refer to the supplementary material available in an online appendix, available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1142/dc1 . Antibodies against amp kinase (ampk), phosporylated ampk (pampk), acetyl coa carboxylase (acc), phosporylated acc, akt, phosporylated akt, and tubulin were from cell signaling; anti - oxphos was from mitosciences; anti cd36-[hrp] was purchased from novus biologicals, and human recombinant insulin (novolin) from novo nordisk canada . This study was performed with the approval of the university of alberta animal policy and welfare committee . Experiments were carried out on male wild - type (c57bl6) and cd36 knockout (ko) mice (19) maintained in a temperature - controlled room with a reversed 12-h light/12-h dark cycle . Mice were left relatively undisturbed for either 1214 or 5258 weeks of age with free access to water and standard rodent diet (category no . 5001; labdiet). At 3234 weeks of age, a subset of mice was randomly divided into a low - fat diet group (category no . D12450b; research diets) and a high - fat diet group (category no . Indirect calorimetry was performed using the comprehensive lab animal monitoring system (oxymax / clams; columbus instruments, colombus, oh). Following an initial 24-h acclimatization period, mice were monitored every 13 min for 24 h to complete a 12-h dark (active)/12-h light (inactive) cycle . The respiratory exchange ratio (rer = vco2/vo2) was used to estimate the percent contribution of fat and carbohydrate to whole - body energy metabolism in mice in vivo . Total activity was calculated by adding z counts (rearing or jumping) to total counts associated with ambulatory movement and stereotypical behavior (grooming and scratching). Upon phospholipid digestion with phospholipase c (2 h at 30c) and lipid extraction, levels of triglycerides were determined in gastrocnemius muscle lysates by gas - liquid chromatography as previously described (20). Identification and quantification of the major long - chain acyl - coa molecular species (c16:0, c18:0, and c18:1) and c18-ceramides were performed by high - performance liquid chromatography as previously described (21). Overnight - fasted mice were anesthetized with sodium pentobarbital, and gastrocnemius muscle was rapidly removed and freeze - clamped in liquid nitrogen and stored at 80c . Acylcarnitine measurements were made using flow - injection tandem mass spectrometry as previously described (14), and organic acids were quantified as previously described (22). Comparisons between groups were performed using unpaired student's two - tailed t test or anova with a bonferroni post hoc test of pairwise comparisons where appropriate . A probability value of <0.05 is considered significant . For further descriptions of the materials and methods, refer to the supplementary material available in an online appendix, available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1142/dc1 . To determine whether an age - related decline in resting metabolic rate and energy expenditure might contribute to the development of insulin resistance, c57bl6 mice of 1214 (young) or 5258 (middle - aged) weeks of age were analyzed using indirect calorimetry . As mice aged and body weight was increased (fig . 1a), substrate use was altered slightly, with reductions in rer indicating that middle - aged mice used more fatty acid throughout the day compared with young mice (fig . In addition, significant reductions in oxygen consumption (vo2) (fig . 1c) and carbon dioxide production (vco2) (fig . 1d) were observed during both the dark (active) and light (inactive) phases in middle - aged mice compared with young mice . 1e) was decreased in middle - aged mice compared with young mice, whereas activity measurements were not significantly different between age - groups (fig . Our data indicate that middle - aged mice have a lower metabolic rate than young mice and that this might increase susceptibility to weight gain, obesity, and metabolic disease . Body weight of young (1214 weeks of age) and middle - aged (5258 weeks of age) mice fed a standard laboratory diet (a). Indirect calorimetry was performed to measure respiratory exchange ratio (rer) (b), vo2 (c), vco2 (d), and heat production adjusted for bodyweight (e); total activity (f) was measured in both the dark (active) and light (inactive) phases . P <0.05 indicates comparisons performed between young and middle - aged mice in either dark or light phases (mann - whitney u test). Two - way anova was performed for rer (b) and indicated the main effect for age (p <0.01). Given that skeletal muscle metabolism contributes to whole - body basal metabolic rate, we addressed whether skeletal muscle metabolism was depressed in middle - aged mice by assessing the activities of two enzymes involved in regulating mitochondrial metabolism . 2a) was elevated in the middle - aged compared with the young mice, and citrate synthase (fig . 2b) followed a similar upward trend, suggesting that -oxidation and tca cycle activity were not directly compromised in the middle - aged mice . Since mitochondrial number (fig . 2c) or function did not appear to be altered at middle age, we next assessed whether peroxisome proliferator activated receptor (ppar) responsive genes or molecular signaling cascades known to regulate skeletal muscle fatty acid flux and fatty acid entry into the mitochondria were associated with this overall reduction in whole - body basal metabolic rate observed in middle age . Although a more comprehensive assessment of the multiple mediators of fatty acid utilization may reveal additional mechanisms, we did not observe any changes in protein levels of known ppar responsive genes involved in lipid metabolism, including malonyl - coa decarboxylase and acyl - coa synthetase 1 (data not shown). As a result, we also examined the energy - sensing kinase, ampk, which is known for its ability to govern energy metabolism (23). In agreement with previous reports using older rodents (24,25), levels of phosphorylated ampk were significantly reduced in skeletal muscle of middle - aged mice compared with young mice (fig . Moreover, the phosphorylation status of acetyl coa carboxylase (acc), the downstream target of ampk that indirectly regulates fatty acid entry into the mitochondria and ultimately -oxidation, was significantly decreased in skeletal muscle from middle - aged mice (fig . Although reduced ampk phosphorylation could be the result of impaired activity of upstream ampk kinases or increased ampk phosphatase activity, it is currently unknown which of these contributes to reduced ampk phosphorylation in our model . However, consistent with decreased energy expenditure, increased adiposity, and reduced ampk activity in skeletal muscle from middle - aged mice, the levels of skeletal muscle triglycerides were significantly elevated in middle - aged mice compared with young mice (fig . 2 g). Given that lipid accumulation in skeletal muscle has been proposed in rodents (26) and humans (17,27,28) to be one of the primary causes of skeletal muscle insulin resistance, we next investigated whether glucose tolerance was impaired in middle - aged mice . Despite elevated intramuscular triglycerides (fig . 2 g) and impaired basal and insulin - stimulated akt phosphorylation (fig . 2h and i, respectively) in skeletal muscle of middle - aged mice, whole - body glucose tolerance (fig . 2k) were not different in middle - aged compared with young mice, suggesting that age - induced alterations in skeletal muscle fatty acid handling and increased triglyceride storage precede the development of insulin resistance and metabolic disease . Alterations in fatty acid handling and reduced insulin signaling in skeletal muscle of middle - aged mice do not result in impaired whole - body glucose tolerance . Activity of two mitochondrial enzymes -had (a) and citrate synthase (cs) (b) was determined in gastrocnemius muscle from overnight fasted young (1214 weeks of age) and middle - aged (5258 weeks of age) mice . Immunoblot analysis using a total oxphos complex antibody cocktail was performed in gastrocnemius muscle lysates, and immunoblots were normalized against tubulin as a control for protein loading (c). Phosphorylation status of ampk at threonine 172 (d) and acc at serine 79 (f) was detected using immunoblot analysis with phospho - specific antibodies . Immunoblots were quantified by densitometry and normalized against total protein levels of ampk (e) and acc (f). Triglyceride (tg) levels (g) and phosphorylation status of akt (h) were determined in gastrocnemius muscle from overnight - fasted young and middle - aged mice . Gastrocnemius muscle was collected from a separate group of overnight - fasted young and middle - aged mice following intraperitoneal injection with either saline or human recombinant insulin (10 units / kg), and immunoblots were performed to detect phosphorylation status of akt at serine 473 in gastrocnemius muscle lysates (i). Glucose tolerance test (j) was performed in young () and middle - aged () mice following a 6-h fast . Serum insulin was detected in young and middle - aged mice after an overnight fast (k). P <0.05 indicates comparisons performed between young and middle - aged mice (mann - whitney u test). Since we speculated that middle - aged mice are more susceptible than young mice to the development of insulin resistance, young and middle - aged mice were subjected to high - fat feeding for 12 weeks . Although young mice fed a high - fat diet displayed weight gain (fig . 3a) and signs of glucose intolerance compared with young mice fed a low - fat diet (fig . In contrast, middle - aged mice fed a high - fat diet showed significant weight gain (body weight: low fat, 30.18 0.70 g, high fat, 55.14 1.56 g; p <0.05) and displayed dramatically elevated insulin levels (fig . In addition, whole - body glucose tolerance in the middle - aged mice fed a high - fat diet was significantly impaired compared with that in middle - aged mice fed a low - fat diet (fig . Although activation of insulin signaling, as determined by the phosphorylation status of akt is not impaired in skeletal muscle of young (fig . 3j) mice fed a high - fat diet compared with that in mice fed a low - fat diet, this is likely due to elevated levels of circulating insulin (fig . 3e and f) observed in the respective high - fat groups . In support of the glucose tolerance data, homeostasis model assessment of insulin resistance values were significantly higher in the high - fat fed middle - aged mice (fig . 3k), suggesting that high - fat feeding induces more dramatic insulin resistance in middle - aged mice than in young mice . Interestingly, young mice on a high - fat diet were of the same weight as middle - aged mice on a low - fat diet; yet, only the young mice on a high - fat diet had impaired glucose disposal (data not shown). Although we cannot discriminate between the effects of aging and increased adiposity because these variables coassociate, our findings do suggest that the increased weight gain associated with aging is not sufficient to alter whole - body glucose disposal and that other factors such as high - fat diet are likely involved . Aging increases the susceptibility to the development of glucose intolerance and insulin resistance in mice fed a high - fat diet . Body weights (a), fed and fasted blood glucose (b), glucose tolerance test (c), and phosphorylation status of akt (d) were measured in gastrocnemius muscle from young (1214 weeks of age) mice following 12 weeks of high fat (hf) feeding . Serum insulin levels in the fasted (e) and fed (f) states obtained from young (1214 weeks of age) and middle - aged (4852 weeks of age) mice fed a low - fat (lf) and high - fat diet for 12 weeks . Fed and fasted blood glucose levels (g), glucose tolerance test (h), area under the curve (auc) for the glucose tolerance test (i), and phosphorylation status of akt in gastrocnemius muscle (j) in middle - aged mice following 12 weeks of high - fat feeding . Homeostasis model assessment of insulin resistance (homa - ir) as a surrogate marker of insulin resistance in young and middle - aged mice fed a high - fat diet (k). Immunoblot analysis using anti - cd36 antibody was performed in gastrocnemius muscle lysates from middle - aged mice fed a low- and high - fat diet, and immunoblots were quantified by densitometry and normalized against tubulin as a control for protein loading (l). Triglyceride (tg) (m), long - chain acyl - coa (lccoa) (n), and c18 ceramide (o) levels in gastrocnemius muscle from overnight - fasted middle - aged mice fed a low- or high - fat diet . Two - way anova was performed to detect main effects of age, diet, and age diet interactions on insulin levels (e and f) and homa - ir (k). Significant effect of age, diet, and age diet interaction (p <0.05) was observed for e, f, and k. * p <0.05 indicates comparisons performed between young and middle - aged mice or between low- and high - fat fed mice in fed or fasted state (mann - whitney u test). Because high - fat diet induced insulin resistance in young rodents is associated with an increased efficiency of fatty acid uptake into skeletal muscle (26), protein expression of cd36, a protein that facilitates fatty acid transport, was determined in skeletal muscle of mice fed a high - fat diet . Consistent with previous publications in young mice (26,29,30), we observed a modest increase in cd36 protein expression in the muscle of young mice fed a high - fat diet, as well as an increase in intramuscular triglyceride levels (data not shown). Consistent with our hypothesis, cd36 expression was significantly elevated in skeletal muscle of middle - aged mice fed a high - fat diet compared with that in mice fed a low - fat diet (fig . Increased cd36 protein expression, skeletal muscle triglyceride levels were significantly elevated in high - fat fed middle - aged mice compared with those in low - fat fed mice (fig . 3 m), as were long - chain acyl - coa esters (fig ., these data suggest that increased cd36-mediated fatty acid transport may contribute to lipid accumulation and impaired insulin sensitivity in skeletal muscle of middle - aged mice fed a high - fat diet . To investigate whether inhibition of fatty acid transport into the skeletal muscle could alter the observed responses of a middle - aged mouse to a high - fat diet, we utilized the cd36 ko mouse, which has skeletal muscle fatty acid uptake rates 4070% of those in wild - type mice (31,32). Interestingly, there was a striking difference in weight gain between middle - aged wild - type and cd36 ko mice following 12 weeks of high - fat feeding (fig . 4a), with middle - aged cd36 ko mice accumulating 51% less weight than the wild - type mice over the same period of time (fig . 4d) between groups could not account for this dramatic change in weight gain, indirect calorimetry indicated that energy expenditure (fig . 4 g) were significantly increased in middle - aged cd36 ko mice fed a high - fat diet compared with those in high - fat fed middle - aged wild - type mice . Although this increased activity in the middle - aged cd36 ko mouse fed a high - fat diet could be attributed to the absence of obesity, heat production was also increased in middle - aged cd36 ko mice fed a low - fat diet compared with that in low - fat fed middle - aged wild - type mice (fig . 4h) and in high - fat fed middle - aged ko mice when normalized for body weight (fig . 4i). Protection of diet - induced obesity in middle - aged cd36 ko mice fed a high - fat (hf) diet for 12 weeks . Representative image of middle - aged (4852 weeks of age) wild - type () and cd36 ko () mice fed a high - fat diet for 12 weeks (a). Weight gain (b) and food intake adjusted for body weight (bw) (c) in wild - type and ko mice fed a high - fat diet . Respiratory exchange ratio (rer) (d), oxygen consumption (vo2) (e), and carbon dioxide production (vco2) (f) in both the dark (active) and light (inactive) phase following 12 weeks of high - fat feeding in wild - type and ko mice . Total activity for a complete dark / light cycle (g), heat production (h), and heat production adjusted for body weight (i) in wild - type and ko mice following 12 weeks of high - fat feeding . Values are the means sem of n = 610 mice in each group . * p <0.05 indicates comparisons performed between the low - fat (lf)-fed mice and between high - fat fed mice (mann - whitney u test [b and g] or anova with bonferroni post hoc test for pairwise comparison). (a high - quality digital representation of this figure is available in the online issue .) To gain a more comprehensive metabolic assessment of muscle metabolism in middle - aged wild - type and cd36 ko mice fed a low - fat or high - fat diet, we used mass spectrometry to measure a broad range of intermediary metabolites, including acylcarnitines of various chain lengths, organic acids, and amino acids . Acylcarnitines are by - products of fuel catabolism that respond to changes in substrate availability or flux limitations at specific mitochondrial enzymes (14,33,34). Middle - aged cd36 ko mice fed a low - fat diet had elevated levels of acetyl - carnitine (c2), and -hydroxybutyryl - carnitine (c4oh) compared with their wild - type counterparts (supplemental fig . 1a and supplemental table 1). Whereas several short - chain acylcarnitine species, including c2 and c4oh, as well as propionyl - carnitine (c3) and succinyl - carnitine (c4dc), tended to increase in response to a high - fat diet, these same metabolites trended downward in cd36 ko mice fed a high - fat diet (supplemental fig . 1a and supplemental table 1). In addition, several long - chain acylcarnitine species were reduced in muscle from wild - type mice fed a high - fat diet while at the same time levels of hydroxylated long - chain acylcarnitine (lcoh) species were increased, resulting in a robust increase in the long - chain to lcoh acylcarnitine ratio (fig . C). Given that long - chain acylcarnitines accumulate when their production by mitochondrial carnitine palmitoyl transferase (cpt)1 exceeds flux through -oxidation enzymes, such as long - chain acyl - coa dehydrogenase (lcad) and -had (35), this pattern is consistent with a diet - induced shift in flux limitation from lcad to -had . Notably, levels of many long - chain and lcoh acylcarnitines were lower in the cd36 ko mice fed a high - fat diet than in the wild - type mice fed a high - fat diet (fig . 5a; supplemental table 1). The organic acids were less responsive to both diet and genotype, although subtle changes were detected in succinate, fumarate, and citrate levels (supplemental fig . Muscle levels of amino acids were higher in wild - type mice fed a low - fat diet but were dramatically decreased following high - fat feeding . By comparison, amino acid levels remained unchanged in cd36 ko mice in response to a high - fat diet (supplemental fig . Although these metabolite measurements do not fully characterize mitochondrial substrate flux, together the data suggest that ablation of cd36 not only alters baseline mitochondrial and intermediary metabolism but also significantly impacts the muscle response to lipid exposure . Altered skeletal muscle lipid handling prevents development of insulin resistance in middle - aged cd36 ko mice fed a high - fat (hf) diet for 12 weeks . Acylcarnitine levels were measured in gastrocnemius muscle from overnight - fasted middle - aged (4852 weeks of age) wild - type (wt) and cd36 ko mice fed a low- (lf) or high - fat diet for 12 weeks . Levels of individual long chain (lc) and lcoh species (a), the sum total of all long - chain or lcoh species (b), and the ratio of total lcoh to long - chain species (c). Phosphorylation status of ampk (thr172) (d) and acc (ser 79) (e) was detected in gastrocnemius muscle using immunoblot analysis . Immunoblots were quantified by densitometry and normalized against total ampk (d) and acc (e). Serum levels of free fatty acids (ffas) from high - fat fed wild - type and cd36 ko mice were determined after 12 weeks of diet (f). Intramuscular levels of triglyceride (tg) (g), lccoa (h), and c18 ceramide (i) were determined in gastrocnemius muscle obtained from wild - type and ko mice fed a high - fat diet for 12 weeks . Glucose tolerance testing (j) was performed in high - fat fed wild - type and ko mice fasted for 6 h. fasted blood glucose (k) and serum insulin (l) levels obtained from middle - aged wild - type and ko mice fed a high - fat diet for 12 weeks . Insulin tolerance test with blood glucose levels expressed as percent change of blood glucose at time zero in middle - aged wild - type and ko mice fed a high - fat diet for 12 weeks (m). Immunoblots were performed on gastrocnemius muscle isolated from middle - aged wild - type and ko mice following high - fat diet and phosphorylation status of akt measured and normalized to total akt levels (n). Main effects of genotype, diet, and genotype diet interactions on acylcarnitine levels (a c) were detected by two - way anova . For simplicity, symbols indicate metabolites that were affected by genotype, diet, or a genotype - diet interaction . Detailed results of the statistical analysis for all acylcarnitine species are presented in supplemental table 1 . * p <0.05 indicates comparisons performed between low - fat fed wild - type and low - fat fed ko mice and between high - fat fed wild - type and high - fat fed ko mice (mann - whitney u test or anova with bonferroni post hoc test [j and m]). Interestingly, despite the changes in muscle acylcarnitine levels, the activity of -had was not altered (data not shown) and middle - aged cd36 ko mice fed a high - fat diet were not protected from decreased levels of ampk or acc phosphorylation (fig . 5d and e) compared with young wild - type mice (fig . 2d and f), suggesting that alternate mechanisms are responsible for the metabolic phenotype observed in cd36 ko mice . Similarly, absolute expression of ampk and acc in skeletal muscle from aged cd36 mice is not different between groups (data not shown). Although serum free fatty acid levels were elevated in high - fat fed cd36 ko mice (fig . 5f), cd36 ablation resulted in a significant reduction in skeletal muscle triglycerides (fig . By contrast, ceramide levels remained similar between high - fat fed groups (fig . 5i), suggesting that accumulation of lipid - derived intermediates other than ceramides, may contribute to impaired insulin sensitivity . Indeed, reduced intramuscular lipid accumulation in middle - aged cd36 ko mice was associated with both improved whole - body glucose tolerance (fig . 5k) compared to high - fat fed middle - aged wild - type mice . Moreover, fasted insulin levels were significantly reduced (fig . 5 m) in high - fat fed middle - aged cd36 ko mice compared to high - fat fed middle - aged wild - type mice, suggesting that insulin sensitivity is restored by preventing lipid accumulation in skeletal muscle . Interestingly, despite improved glucose utilization and reduced plasma insulin levels in these mice, the phosphorylation status of akt was similar in skeletal muscle from middle - aged wild - type and cd36 ko mice on high - fat diet (fig . Furthermore, we found no difference in glycogen content in livers from cd36 ko mice fed a low - fat or high - fat diet (data not shown), suggesting that the improved glucose tolerance observed in these mice is the result of increased glucose uptake and/or glucose oxidation in skeletal muscle from cd36 ko mice and not alterations in hepatic glucose metabolism . To determine whether an age - related decline in resting metabolic rate and energy expenditure might contribute to the development of insulin resistance, c57bl6 mice of 1214 (young) or 5258 (middle - aged) weeks of age were analyzed using indirect calorimetry . As mice aged and body weight was increased (fig . 1a), substrate use was altered slightly, with reductions in rer indicating that middle - aged mice used more fatty acid throughout the day compared with young mice (fig . In addition, significant reductions in oxygen consumption (vo2) (fig . 1c) and carbon dioxide production (vco2) (fig . 1d) were observed during both the dark (active) and light (inactive) phases in middle - aged mice compared with young mice . 1e) was decreased in middle - aged mice compared with young mice, whereas activity measurements were not significantly different between age - groups (fig . Our data indicate that middle - aged mice have a lower metabolic rate than young mice and that this might increase susceptibility to weight gain, obesity, and metabolic disease . Body weight of young (1214 weeks of age) and middle - aged (5258 weeks of age) mice fed a standard laboratory diet (a). Indirect calorimetry was performed to measure respiratory exchange ratio (rer) (b), vo2 (c), vco2 (d), and heat production adjusted for bodyweight (e); total activity (f) was measured in both the dark (active) and light (inactive) phases . P <0.05 indicates comparisons performed between young and middle - aged mice in either dark or light phases (mann - whitney u test). Two - way anova was performed for rer (b) and indicated the main effect for age (p <0.01). Given that skeletal muscle metabolism contributes to whole - body basal metabolic rate, we addressed whether skeletal muscle metabolism was depressed in middle - aged mice by assessing the activities of two enzymes involved in regulating mitochondrial metabolism . 2a) was elevated in the middle - aged compared with the young mice, and citrate synthase (fig . 2b) followed a similar upward trend, suggesting that -oxidation and tca cycle activity were not directly compromised in the middle - aged mice . Since mitochondrial number (fig . 2c) or function did not appear to be altered at middle age, we next assessed whether peroxisome proliferator activated receptor (ppar) responsive genes or molecular signaling cascades known to regulate skeletal muscle fatty acid flux and fatty acid entry into the mitochondria were associated with this overall reduction in whole - body basal metabolic rate observed in middle age . Although a more comprehensive assessment of the multiple mediators of fatty acid utilization may reveal additional mechanisms, we did not observe any changes in protein levels of known ppar responsive genes involved in lipid metabolism, including malonyl - coa decarboxylase and acyl - coa synthetase 1 (data not shown). As a result, we also examined the energy - sensing kinase, ampk, which is known for its ability to govern energy metabolism (23). In agreement with previous reports using older rodents (24,25), levels of phosphorylated ampk were significantly reduced in skeletal muscle of middle - aged mice compared with young mice (fig . Moreover, the phosphorylation status of acetyl coa carboxylase (acc), the downstream target of ampk that indirectly regulates fatty acid entry into the mitochondria and ultimately -oxidation, was significantly decreased in skeletal muscle from middle - aged mice (fig . Although reduced ampk phosphorylation could be the result of impaired activity of upstream ampk kinases or increased ampk phosphatase activity, it is currently unknown which of these contributes to reduced ampk phosphorylation in our model . However, consistent with decreased energy expenditure, increased adiposity, and reduced ampk activity in skeletal muscle from middle - aged mice, the levels of skeletal muscle triglycerides were significantly elevated in middle - aged mice compared with young mice (fig . 2 g). Given that lipid accumulation in skeletal muscle has been proposed in rodents (26) and humans (17,27,28) to be one of the primary causes of skeletal muscle insulin resistance, we next investigated whether glucose tolerance was impaired in middle - aged mice . Despite elevated intramuscular triglycerides (fig . 2 g) and impaired basal and insulin - stimulated akt phosphorylation (fig . 2h and i, respectively) in skeletal muscle of middle - aged mice, whole - body glucose tolerance (fig . 2k) were not different in middle - aged compared with young mice, suggesting that age - induced alterations in skeletal muscle fatty acid handling and increased triglyceride storage precede the development of insulin resistance and metabolic disease . Alterations in fatty acid handling and reduced insulin signaling in skeletal muscle of middle - aged mice do not result in impaired whole - body glucose tolerance . Activity of two mitochondrial enzymes -had (a) and citrate synthase (cs) (b) was determined in gastrocnemius muscle from overnight fasted young (1214 weeks of age) and middle - aged (5258 weeks of age) mice . Immunoblot analysis using a total oxphos complex antibody cocktail was performed in gastrocnemius muscle lysates, and immunoblots were normalized against tubulin as a control for protein loading (c). Phosphorylation status of ampk at threonine 172 (d) and acc at serine 79 (f) was detected using immunoblot analysis with phospho - specific antibodies . Immunoblots were quantified by densitometry and normalized against total protein levels of ampk (e) and acc (f). Triglyceride (tg) levels (g) and phosphorylation status of akt (h) were determined in gastrocnemius muscle from overnight - fasted young and middle - aged mice . Gastrocnemius muscle was collected from a separate group of overnight - fasted young and middle - aged mice following intraperitoneal injection with either saline or human recombinant insulin (10 units / kg), and immunoblots were performed to detect phosphorylation status of akt at serine 473 in gastrocnemius muscle lysates (i). Glucose tolerance test (j) was performed in young () and middle - aged () mice following a 6-h fast . Serum insulin was detected in young and middle - aged mice after an overnight fast (k). P <0.05 indicates comparisons performed between young and middle - aged mice (mann - whitney u test). Since we speculated that middle - aged mice are more susceptible than young mice to the development of insulin resistance, young and middle - aged mice were subjected to high - fat feeding for 12 weeks . 3a) and signs of glucose intolerance compared with young mice fed a low - fat diet (fig . In contrast, middle - aged mice fed a high - fat diet showed significant weight gain (body weight: low fat, 30.18 0.70 g, high fat, 55.14 1.56 g; p <0.05) and displayed dramatically elevated insulin levels (fig . In addition, whole - body glucose tolerance in the middle - aged mice fed a high - fat diet was significantly impaired compared with that in middle - aged mice fed a low - fat diet (fig . Although activation of insulin signaling, as determined by the phosphorylation status of akt is not impaired in skeletal muscle of young (fig . 3j) mice fed a high - fat diet compared with that in mice fed a low - fat diet, this is likely due to elevated levels of circulating insulin (fig . 3e and f) observed in the respective high - fat groups . In support of the glucose tolerance data, homeostasis model assessment of insulin resistance values were significantly higher in the high - fat fed middle - aged mice (fig . 3k), suggesting that high - fat feeding induces more dramatic insulin resistance in middle - aged mice than in young mice . Interestingly, young mice on a high - fat diet were of the same weight as middle - aged mice on a low - fat diet; yet, only the young mice on a high - fat diet had impaired glucose disposal (data not shown). Although we cannot discriminate between the effects of aging and increased adiposity because these variables coassociate, our findings do suggest that the increased weight gain associated with aging is not sufficient to alter whole - body glucose disposal and that other factors such as high - fat diet are likely involved . Aging increases the susceptibility to the development of glucose intolerance and insulin resistance in mice fed a high - fat diet . Body weights (a), fed and fasted blood glucose (b), glucose tolerance test (c), and phosphorylation status of akt (d) were measured in gastrocnemius muscle from young (1214 weeks of age) mice following 12 weeks of high fat (hf) feeding . Serum insulin levels in the fasted (e) and fed (f) states obtained from young (1214 weeks of age) and middle - aged (4852 weeks of age) mice fed a low - fat (lf) and high - fat diet for 12 weeks . Fed and fasted blood glucose levels (g), glucose tolerance test (h), area under the curve (auc) for the glucose tolerance test (i), and phosphorylation status of akt in gastrocnemius muscle (j) in middle - aged mice following 12 weeks of high - fat feeding . Homeostasis model assessment of insulin resistance (homa - ir) as a surrogate marker of insulin resistance in young and middle - aged mice fed a high - fat diet (k). Immunoblot analysis using anti - cd36 antibody was performed in gastrocnemius muscle lysates from middle - aged mice fed a low- and high - fat diet, and immunoblots were quantified by densitometry and normalized against tubulin as a control for protein loading (l). Triglyceride (tg) (m), long - chain acyl - coa (lccoa) (n), and c18 ceramide (o) levels in gastrocnemius muscle from overnight - fasted middle - aged mice fed a low- or high - fat diet . Two - way anova was performed to detect main effects of age, diet, and age diet interactions on insulin levels (e and f) and homa - ir (k). Significant effect of age, diet, and age diet interaction (p <0.05) was observed for e, f, and k. * p <0.05 indicates comparisons performed between young and middle - aged mice or between low- and high - fat fed mice in fed or fasted state (mann - whitney u test). Because high - fat diet induced insulin resistance in young rodents is associated with an increased efficiency of fatty acid uptake into skeletal muscle (26), protein expression of cd36, a protein that facilitates fatty acid transport, was determined in skeletal muscle of mice fed a high - fat diet . Consistent with previous publications in young mice (26,29,30), we observed a modest increase in cd36 protein expression in the muscle of young mice fed a high - fat diet, as well as an increase in intramuscular triglyceride levels (data not shown). Consistent with our hypothesis, cd36 expression was significantly elevated in skeletal muscle of middle - aged mice fed a high - fat diet compared with that in mice fed a low - fat diet (fig . 3l). In accordance with increased cd36 protein expression, skeletal muscle triglyceride levels were significantly elevated in high - fat fed middle - aged mice compared with those in low - fat fed mice (fig . 3 m), as were long - chain acyl - coa esters (fig . Together, these data suggest that increased cd36-mediated fatty acid transport may contribute to lipid accumulation and impaired insulin sensitivity in skeletal muscle of middle - aged mice fed a high - fat diet . To investigate whether inhibition of fatty acid transport into the skeletal muscle could alter the observed responses of a middle - aged mouse to a high - fat diet, we utilized the cd36 ko mouse, which has skeletal muscle fatty acid uptake rates 4070% of those in wild - type mice (31,32). Interestingly, there was a striking difference in weight gain between middle - aged wild - type and cd36 ko mice following 12 weeks of high - fat feeding (fig . 4a), with middle - aged cd36 ko mice accumulating 51% less weight than the wild - type mice over the same period of time (fig . 4d) between groups could not account for this dramatic change in weight gain, indirect calorimetry indicated that energy expenditure (fig . 4 g) were significantly increased in middle - aged cd36 ko mice fed a high - fat diet compared with those in high - fat fed middle - aged wild - type mice . Although this increased activity in the middle - aged cd36 ko mouse fed a high - fat diet could be attributed to the absence of obesity, heat production was also increased in middle - aged cd36 ko mice fed a low - fat diet compared with that in low - fat fed middle - aged wild - type mice (fig . 4h) and in high - fat fed middle - aged ko mice when normalized for body weight (fig . 4i). Protection of diet - induced obesity in middle - aged cd36 ko mice fed a high - fat (hf) diet for 12 weeks . Representative image of middle - aged (4852 weeks of age) wild - type () and cd36 ko () mice fed a high - fat diet for 12 weeks (a). Weight gain (b) and food intake adjusted for body weight (bw) (c) in wild - type and ko mice fed a high - fat diet . Respiratory exchange ratio (rer) (d), oxygen consumption (vo2) (e), and carbon dioxide production (vco2) (f) in both the dark (active) and light (inactive) phase following 12 weeks of high - fat feeding in wild - type and ko mice . Total activity for a complete dark / light cycle (g), heat production (h), and heat production adjusted for body weight (i) in wild - type and ko mice following 12 weeks of high - fat feeding . * p <0.05 indicates comparisons performed between the low - fat (lf)-fed mice and between high - fat fed mice (mann - whitney u test [b and g] or anova with bonferroni post hoc test for pairwise comparison). (a high - quality digital representation of this figure is available in the online issue .) To gain a more comprehensive metabolic assessment of muscle metabolism in middle - aged wild - type and cd36 ko mice fed a low - fat or high - fat diet, we used mass spectrometry to measure a broad range of intermediary metabolites, including acylcarnitines of various chain lengths, organic acids, and amino acids . Acylcarnitines are by - products of fuel catabolism that respond to changes in substrate availability or flux limitations at specific mitochondrial enzymes (14,33,34). Middle - aged cd36 ko mice fed a low - fat diet had elevated levels of acetyl - carnitine (c2), and -hydroxybutyryl - carnitine (c4oh) compared with their wild - type counterparts (supplemental fig . 1a and supplemental table 1). Whereas several short - chain acylcarnitine species, including c2 and c4oh, as well as propionyl - carnitine (c3) and succinyl - carnitine (c4dc), tended to increase in response to a high - fat diet, these same metabolites trended downward in cd36 ko mice fed a high - fat diet (supplemental fig . 1a and supplemental table 1). In addition, several long - chain acylcarnitine species were reduced in muscle from wild - type mice fed a high - fat diet while at the same time levels of hydroxylated long - chain acylcarnitine (lcoh) species were increased, resulting in a robust increase in the long - chain to lcoh acylcarnitine ratio (fig . 5a c). Given that long - chain acylcarnitines accumulate when their production by mitochondrial carnitine palmitoyl transferase (cpt)1 exceeds flux through -oxidation enzymes, such as long - chain acyl - coa dehydrogenase (lcad) and -had (35), this pattern is consistent with a diet - induced shift in flux limitation from lcad to -had . Notably, levels of many long - chain and lcoh acylcarnitines were lower in the cd36 ko mice fed a high - fat diet than in the wild - type mice fed a high - fat diet (fig . The organic acids were less responsive to both diet and genotype, although subtle changes were detected in succinate, fumarate, and citrate levels (supplemental fig . Muscle levels of amino acids were higher in wild - type mice fed a low - fat diet but were dramatically decreased following high - fat feeding . By comparison, amino acid levels remained unchanged in cd36 ko mice in response to a high - fat diet (supplemental fig . Although these metabolite measurements do not fully characterize mitochondrial substrate flux, together the data suggest that ablation of cd36 not only alters baseline mitochondrial and intermediary metabolism but also significantly impacts the muscle response to lipid exposure . Altered skeletal muscle lipid handling prevents development of insulin resistance in middle - aged cd36 ko mice fed a high - fat (hf) diet for 12 weeks . Acylcarnitine levels were measured in gastrocnemius muscle from overnight - fasted middle - aged (4852 weeks of age) wild - type (wt) and cd36 ko mice fed a low- (lf) or high - fat diet for 12 weeks . Levels of individual long chain (lc) and lcoh species (a), the sum total of all long - chain or lcoh species (b), and the ratio of total lcoh to long - chain species (c). Phosphorylation status of ampk (thr172) (d) and acc (ser 79) (e) was detected in gastrocnemius muscle using immunoblot analysis . Immunoblots were quantified by densitometry and normalized against total ampk (d) and acc (e). Serum levels of free fatty acids (ffas) from high - fat fed wild - type and cd36 ko mice were determined after 12 weeks of diet (f). Intramuscular levels of triglyceride (tg) (g), lccoa (h), and c18 ceramide (i) were determined in gastrocnemius muscle obtained from wild - type and ko mice fed a high - fat diet for 12 weeks . Glucose tolerance testing (j) was performed in high - fat fed wild - type and ko mice fasted for 6 h. fasted blood glucose (k) and serum insulin (l) levels obtained from middle - aged wild - type and ko mice fed a high - fat diet for 12 weeks . Insulin tolerance test with blood glucose levels expressed as percent change of blood glucose at time zero in middle - aged wild - type and ko mice fed a high - fat diet for 12 weeks (m). Immunoblots were performed on gastrocnemius muscle isolated from middle - aged wild - type and ko mice following high - fat diet and phosphorylation status of akt measured and normalized to total akt levels (n). Main effects of genotype, diet, and genotype diet interactions on acylcarnitine levels (a c) were detected by two - way anova . For simplicity, symbols indicate metabolites that were affected by genotype, diet, or a genotype - diet interaction . Detailed results of the statistical analysis for all acylcarnitine species are presented in supplemental table 1 . * p <0.05 indicates comparisons performed between low - fat fed wild - type and low - fat fed ko mice and between high - fat fed wild - type and high - fat fed ko mice (mann - whitney u test or anova with bonferroni post hoc test [j and m]). Interestingly, despite the changes in muscle acylcarnitine levels, the activity of -had was not altered (data not shown) and middle - aged cd36 ko mice fed a high - fat diet were not protected from decreased levels of ampk or acc phosphorylation (fig . 5d and e) compared with young wild - type mice (fig . 2d and f), suggesting that alternate mechanisms are responsible for the metabolic phenotype observed in cd36 ko mice . Similarly, absolute expression of ampk and acc in skeletal muscle from aged cd36 mice is not different between groups (data not shown). Although serum free fatty acid levels were elevated in high - fat fed cd36 ko mice (fig . 5f), cd36 ablation resulted in a significant reduction in skeletal muscle triglycerides (fig . By contrast, ceramide levels remained similar between high - fat fed groups (fig . 5i), suggesting that accumulation of lipid - derived intermediates other than ceramides, may contribute to impaired insulin sensitivity . Indeed, reduced intramuscular lipid accumulation in middle - aged cd36 ko mice was associated with both improved whole - body glucose tolerance (fig . 5k) compared to high - fat fed middle - aged wild - type mice . 5 m) in high - fat fed middle - aged cd36 ko mice compared to high - fat fed middle - aged wild - type mice, suggesting that insulin sensitivity is restored by preventing lipid accumulation in skeletal muscle . Interestingly, despite improved glucose utilization and reduced plasma insulin levels in these mice, the phosphorylation status of akt was similar in skeletal muscle from middle - aged wild - type and cd36 ko mice on high - fat diet (fig . Furthermore, we found no difference in glycogen content in livers from cd36 ko mice fed a low - fat or high - fat diet (data not shown), suggesting that the improved glucose tolerance observed in these mice is the result of increased glucose uptake and/or glucose oxidation in skeletal muscle from cd36 ko mice and not alterations in hepatic glucose metabolism . Consistent with previous reports (17), our data show a significant decline in metabolic rate in middle - aged mice when compared to their young counterparts (fig . Interestingly, although mitochondrial function was not directly assessed in this study, the decline in overall metabolic rate did not appear to stem from compromised mitochondrial function in skeletal muscle of middle - aged mice compared to young mice . Indeed, whole body rer was modestly decreased with aging and muscle activity of -had increased, suggesting there is a shift in substrate selection from carbohydrates toward fatty acids . However, the age - associated reduction in overall metabolic rate did not appear to correlate with changes in maximal activities of mitochondrial enzymes in muscle from young and middle - aged mice (fig . 2a and 2b), suggesting other factors may influence substrate oxidation in muscle, as well as overall metabolic rate . Consistent with this, the ampk / acc signaling axis was significantly reduced in skeletal muscle from middle - aged mice (fig . 2d and 2f). Still unclear is whether reduced ampk phosphorylation in the older mice reflects a cause or consequence of reduced metabolic rate . Although results of a recent study suggest that acc - mediated shifts in fat oxidation per se do not impact whole body energy expenditure or susceptibility to diet - induced obesity (36), ampk acts on a broad range of enzymatic and transcriptional targets that could affect energy balance via mechanisms other than substrate selection (25,37). Nonetheless, cd36 deficiency raised metabolic rate without activating ampk, indicating that other mechanisms are operative in this model (see below). Although akt phosphorylation is reduced in skeletal muscle from middle - aged mice compared to young mice (fig . 2h and 2i) whole - body glucose tolerance and plasma insulin levels remained normal (fig . 2j and 2k), suggesting that impaired activation of insulin - signaling parameters in skeletal muscle precede overt changes in whole body glucose disposal and may increase the susceptibility of aged mice to diet - induced insulin resistance . To determine whether middle - aged mice indeed are more susceptible to developing insulin resistance in response to a high - fat diet compared to their younger counterparts, we subjected middle - aged mice to 12 weeks of a low - fat or high - fat diet . As expected, middle - aged mice gained significantly more weight than young mice when fed a high - fat diet (weight gain: middle - aged, 22.8 1.9 g versus young, 12.0 1.1 g; p <0.01). 3c, 3h and 3i), high - fat diet - induced hyperinsulinemia was only observed in middle - aged mice (fig . 3e and 3f), indicating that increased -cell insulin secretion was sufficient to offset overt hyperglycemia in this age - group (fig . 3 g). Given that prolonged hypersecretion of insulin by the -cells to compensate for peripheral insulin resistance can contribute to -cell failure (38) and potentially type 2 diabetes (39), our data suggest that middle - aged mice have a heightened susceptibility to the development of insulin resistance . However, this susceptibility might not be due to aging per se, since adiposity was also increased in the older mice . As our study design does not permit to discriminate between the effects of aging and adiposity on skeletal muscle metabolism and the development of insulin resistance, future experiments should be directed at conducting weight loss (food restriction) studies or exercise studies to determine if these nevertheless, as aging and increased adiposity co - associate our findings likely reflect the majority of middle - aged humans in the western world who are at risk of developing insulin resistance . Although intramuscular lipid accumulation associated with aging did not appear to result from mitochondrial dysfunction, we do show a significant twofold increase in cd36 protein expression in skeletal muscle of high - fat fed middle - aged mice compared to low - fat fed middle - aged mice (fig . 3l), suggesting fatty acid transport into muscle exceeded the capacity for their oxidation . Although it is unknown what caused increased cd36 expression in our study, it may have resulted from high levels of plasma glucose levels that have been shown to regulate cd36 expression through both transcriptional and/or translational mechanisms in rodents and humans (40,41). To determine whether reduced fatty acid transport and metabolism could rescue the high - fat diet - induced phenotype consistent with our prediction, cd36 deficiency prevented the decline in metabolic rate and energy expenditure in middle - aged mice fed a high - fat diet as compared to age - matched wild - type mice (fig . 4e and 4f). Since food intake was similar between groups (fig . 4c), we propose that increased energy expenditure in high - fat fed middle - aged cd36 ko mice contributes to their protection against diet - induced obesity (fig . 4b). The blunted decline in metabolic rate in the middle - aged cd36 ko mice fed a high - fat diet (fig . 4e and 4f) was accompanied by alterations in muscle concentrations of several metabolic intermediates (fig . 1), but no improvement in ampk and acc phosphorylation (fig . 5d and 5e, respectively). The impact of the diet on muscle metabolites in this study differed to some extent as compared to a previous report (14) in which tissue specimens were harvested from younger animals in the fed state . Herein, tissues were collected after an overnight fast because we sought to evaluate a state of heightened fatty acid oxidation . Under these conditions, the drop in long - chain acylcarnitines could reflect decreased fatty acid availability, lower cpt1 activity or increased long - chain acyl coa flux through lcad . First, the high - fat diet increases rather than decreases lipid delivery to muscle . Second, of the two major products of cpt1 (palmitoylcarnitine (c16) and oleylcarnitine (c18:1), only the unsaturated species was reduced by the diet (fig . 5a), suggesting upregulation of the isomerase enzyme that catalyzes conversion of the double bond (42). 5c) in response to chronic lipid exposure suggest a shift in flux limitation from the earlier to later steps in -oxidation . Lastly, the diet resulted in a robust drop in whole body rer, indicative of a systemic increase in fat oxidation . Notably, cd36 deficiency altered baseline levels of several muscle metabolites, and in general, tended to mitigate diet - induced changes in several acylcarnitine and amino acid species . This apparent resistance to diet - induced metabolic perturbations in the cd36 ko mice might be directly related to a reduction in fat delivery and/or secondary to enhanced energy expenditure and insulin sensitivity . Although further work is necessary to fully understand the implications of these results, it is clear that loss of cd36 has a global impact on muscle fuel metabolism . In addition, it is possible that other organs such as adipose tissue, liver and brain (43,44) are also involved in maintaining a high level of energy expenditure in the middle - aged cd36 ko mice . Overall, ablation of cd36 was associated with an improvement in whole - body glucose utilization and insulin sensitivity (fig . Although the mechanisms responsible for this are not known, excessive intramuscular lipid accumulation induced by high - fat feeding was prevented in skeletal muscle of middle - aged cd36 ko mice (fig . 5 g and 5h). Whereas elevated intramuscular triglyceride levels were not associated with insulin resistance in young mice (fig . 2j2k), preventing the more dramatic age- and diet - induced accumulation of intramuscular triglyceride and long - chain acyl coa levels in middle - aged cd36 ko mice correlated with improved whole - body insulin sensitivity . Although there is ample evidence indicating that cd36 ablation significantly reduces skeletal muscle fatty acid uptake (31,32), it is also possible that the effects that we report using the cd36 ko mice are secondary to changes in fatty acid metabolism . Notwithstanding this later possibility, our data demonstrate that limiting cd36-mediated skeletal muscle fatty acid transport guards against whole - body and muscle insulin resistance in middle - aged mice fed a high - fat diet . This finding suggests a potential therapeutic strategy for combating metabolic disease in the face of age - related abnormalities.

Sarcopenia is present in approximately 513% of persons over the age of 60 years and defined as the loss of skeletal muscle mass and strength with progressive decline in mobility and function . The pathogenesis of sarcopenia is associated with many intrinsic and extrinsic factors, including proinflammatory cytokine accumulation, oxidative stress, mitochondrial dysfunction, insulin resistance, and aging - related loss of anabolic hormones and motor neuron end plates . The loss of skeletal muscle results from an imbalance of protein metabolism, which is the dynamic balance between protein degradation and protein synthesis . The protein degradation systems in skeletal muscle are modulated by a coordinated network of signaling pathways activated or suppressed by hormones and cytokines; therefore, catabolism is stimulated by a variety of proinflammatory cytokines, glucocorticoids, and reactive oxygen species (ros) [5, 6]. It is important to note that impaired cellular immune function combined with low - grade inflammation represents a continuous impact in aging process . Although aging is associated with prolonged inflammatory activity that is mainly attributed to progressively worsening muscle weakness, it is unclear whether these processes are cross - talked . Molecular signals and pathways connecting inflammatory system and muscle degeneration may be the key to reveal the interactions responsible for the progression of sarcopenia . The interplay seems to exist between the mass loss of skeletal muscle and elevated systemic inflammation (figure 1). Sarcopenia is a complex process with a subclinical state of inflammation driven by proinflammatory cytokines and oxidative stress, which increases the infiltration of immune cells into injured muscles . In turn, inflammation aggravates muscle loss and fat accumulation in the aging skeletal muscle and further decreases muscle function and physical activity . The increase in chronic inflammation response associated with high - level proinflammatory mediators as the extension of age has been considered as one of the diagnostic hallmarks and a significant contributor to aging - related atrophy of skeletal muscle . The transcription factor, nuclear factor-b (nf-b), has been considered as an important mediator underlying the relationship between inflammation and aging [10, 11]. The correlation between inflammation and sarcopenia exists as a possible linkage describing the effect of inflammation on the balance between protein anabolism and catabolism, with the presence of cd68 + macrophage infiltration . It is worth noting that substantial evidence also demonstrates the connection between obesity and sarcopenia, namely, sarcopenic obesity . In fact, obesity always plays an important role in sarcopenia, by the way of adding inflammatory burden . During obesity, adipose tissue is characterized by a chronic inflammatory state, through the release of numerous proinflammatory cytokines including tumor necrosis factor - alpha (tnf-), interleukin-6 (il-6), and interleukin-1 beta (il-1) as the factors largely responsible for insulin resistance in obese adipose tissue combined with aging - related skeletal muscle loss . Moreover, impaired mitochondria can be both the reason and the consequence of inflammation during aging . Increasing evidence shows that mitochondria may contribute to inflammation via ros production, nf-b activation, calcium homeostasis, impaired autophagy, and atp deficiency [17, 18]. Dysfunctional mitochondria are able to modulate aging - related inflammatory processes through direct activation of nlrp3 inflammasome, which can correspondingly result in the activation of caspase-1 or redox - sensitive inflammatory signaling pathways, thus leading to the production of il-1 and il-18 [19, 20]. It should be noted that increasing ros could stimulate the activation of nf-b via nf-b - inducing kinase (nik) and ib kinase and (ikk/) [21, 22]. Since the increased redox activation in the presence of transcription factor nf-b, excessive ros generation plays an important role in impaired mitochondrial function and oxidative capacity and accelerates the aging process of skeletal muscle . As described above, sarcopenia is a common feature in the elderly and mainly related to the release of inflammatory mediators from damaged tissue . These responses are controlled by a combination of various cytokines responsible for inflammatory pathways [10, 24]. Given that the inflammatory response is a complex system, cytokines are important not only as the indicators for mediating chronic inflammatory state through increasing protein degradation and reducing protein synthesis, but also as the mediators for controlling muscle wasting by directly targeting muscle tissue . In particular, proinflammatory cytokines are well known to impinge on protein metabolism in skeletal muscle and result in the activation of catabolism signals or upregulate inflammatory pathways such as nf-b and stat3, thus finally leading to the increased activation of ubiquitin - proteasome and autophagy system [26, 27]. The chronic inflammatory aging process depends not only on increased expression of proinflammatory factors, but also on reduced levels of anti - inflammatory factors such as il-10, one of the anti - inflammatory cytokines . Since the presence and function of cytokines have been demonstrated in the pathogenesis of sarcopenia, their origins and types must be identified . In fact, cytokines can be secreted by various types of cells like inflammatory and stromal cells, as well as skeletal muscle cells . In skeletal muscle, the constitutive expression of cytokines is generally stronger in differentiated myotubes compared with myoblasts, which usually results in inconspicuous change in cytokine release to stimuli [29, 30]. Accumulating evidences over the past decade have demonstrated that those proinflammatory cytokines such as tnf-, il-6, and c - reactive protein (crp) cause a significant increase in aging skeletal muscle cells and play a key role in the complex network of inflammatory signals in charge of muscle homeostasis connected with aging - related disability and mortality [31, 32]. Has originally been classified as a prototypical proinflammatory cytokine to exhibit marked pleiotropy, and its anti - inflammatory property has also been identified later [34, 35]. Il-6 plays an important role in the pathogenesis of several chronic diseases including sarcopenia by regulating inflammatory and metabolic functions . Il-6 signaling involves the binding to the membrane - bound il-6 receptor in skeletal muscle and the activation of downstream signaling pathways including stat3, mapk / erk, p38, myostatin, and foxo3 pathways [3740]. Additionally, il-6 has confirmed to have the function of activating ampk and/or phosphatidylinositol-3-kinase (pi3k) and regulating the metabolism in skeletal muscle [41, 42]. The overexpression of il-6 can result in reduced body mass and impaired insulin - stimulated glucose uptake in mouse skeletal muscle . Furthermore, the infusion of il-6 in skeletal muscle can reduce the phosphorylation of s6k1, which is activated by akt / mtor and associated with the inhibition of anabolic process . A comparative analysis of cytokine levels has confirmed the upregulation of proinflammatory il-6 and crp in the elderly, along with increased risk for the loss of skeletal muscle mass and strength . According to a recent study, the serum high - sensitivity crp (hs - crp) levels in the obesity only and in the sarcopenic obesity groups are significantly higher than that in the normal group after multivariate adjustments, which provides the evidence that obesity and sarcopenic obesity are associated with increased levels of serum hs - crp among males . Tnf- is a cytokine implicated in the metabolic disturbance of chronic inflammation, with the formation of il-1, which has been identified as a circulatory factor to increase gluconeogenesis, lipolysis, and proteolysis, accompanied with the decrease in protein, lipid, and glycogen synthesis in skeletal muscle . During the stages of muscle regeneration, tnf- and il-1 are observed in injured muscle with an accumulation of macrophages [48, 49]. Also, tnf- and il-1 have been confirmed to promote il-6 secretion through activating nf-b in cultured skeletal muscle cells . Several previous studies have confirmed that tnf- at the elevated level can increase catabolism in skeletal muscle by suppressing akt / mtor pathway . Tnf- and its soluble receptors have been described as the important contributors or biomarkers for the loss of mass and strength in aged skeletal muscle . There is a strongly negative correlation between protein breakdown and tnf- concentration in the elderly . Injecting tnf- into mice has revealed the activation of ubiquitin - proteasome system and the decrease of skeletal muscle function . In vivo, the synthesis rate of myosin heavy chain protein is correlated negatively with the expression of tnf- in skeletal muscle . Tnf- induces skeletal muscle loss through increased myofibrillar protein degradation and cell apoptosis, thus resulting in muscle atrophy and the inhibition of muscle regeneration following injury [55, 56]. Additionally, it seems that tnf- may antagonize the anabolic effect of insulin growth factor-1 (igf-1), due to the development of growth hormone resistance, which decreases both circulating and muscular igf-1 [57, 58]. Recent studies have shown that g / a-308 tnf- polymorphism as a marker of sarcopenia in normal weight obese syndrome, suggesting the importance of tnf- in the diagnosis of sarcopenia . It is important to note that an increase in tnf- alone is not sufficient to cause muscle atrophy . The upregulation of nf-b can cause muscle atrophy in rodents and contribute to the progressive muscle loss of advancing age . Other mediators such as interferon- (ifn-) are produced in the microenvironment of skeletal muscle and play a critical role during myogenesis . Moreover, il-15 is usually mentioned among paracrine effectors, while cytokines such as irisin and myonectin are able to induce anti - inflammatory cytokines (il-1 receptor antagonist and il-10), especially during contraction and aging conditions . The levels of tlr4 protein and il6, il10, and il15 mrna expression are increased after short - time bed rest in healthy older adults, while the levels of ifn- and macrophage inflammatory protein-1-beta (mip-1) are elevated in aging skeletal muscle . Furthermore, peroxisome proliferator - activated receptor gamma coactivator-1-alpha (pgc-1) is regarded to have an anti - inflammatory function by inhibiting the function of foxo3, which could promote inflammatory cytokine expression and downregulate antioxidant enzyme expression in aging muscle . The reduced expression of pgc-1 results in a low level and a systemic inflammatory response to exhibit negative impacts on skeletal muscle . Inversely, the upregulated pgc-1 can reduce the activity of nf-b, which contributes to the inhibition of proinflammatory cytokines to be benefit for the prevention of mass and strength loss of skeletal muscle and functional decline of other organs, as well as the ultimate impact on homeostasis in human body [68, 69]. Proinflammatory cytokines have a wide variety of roles in inflammation systems and may be the important factor predisposing to muscle catabolism response in the elderly . However, the roles of these cytokines in aging skeletal muscle are still not fully understood . Furthermore, the tissue - specific inflammatory signaling pathways in response to cytokines along with the elevation of systemic cytokines are important elements to be considered . Mirnas are short noncoding rnas with approximately 22 nucleotides in length and are involved in the complex posttranscriptional regulatory networks and the maintenance of healthy cellular functions such as growth, development, and metabolism . Skeletal muscle is the most abundant tissue in human body, comprising 4050% of body mass . It is estimated that approximately 60% of human genes are regulated by mirnas, suggesting that highly enriched mirnas in skeletal muscle play important roles in biological processes by gene silencing, including aging process . The functions of mirnas can be achieved either by suppressing the translation of target messenger rnas (mrnas) or by promoting the degradation of mrnas, thereby providing a powerful and sensitive regulator to tune gene expression and cell functions during the aging process of skeletal muscle . Mirnas, from noncoding rna genes or within protein - coding genes, are transcribed into primicrornas by rna polymerase ii or polymerase iii in some cases and subsequently embedded into premicrorna hairpins like rna duplex by drosha [73, 74]. Premicrorna hairpins are exported from nucleus by exportin-5 and processed into double - stranded mature mirnas by dicer in combination with its rna - binding cofactor . After dicer - mediated maturation, the mirnas orient the risc complex with the removal or the preservation of one strand as the guide strand and preferentially load on the risc complex in position at regulatory sequences in target genes . Although the precise mechanisms for mirna targeting and activity still remain to be fully explored, mirna activity appears to be largely dependent on its binding capacity to the target mrna molecule [7779]. Generally, mrnas contain a predicted binding site in the 3 untranslated region (utr), less commonly, in the 5 utr, and many mrnas contain multiple potential binding sites . According to the binding complementarity of the seed sequence, argonaute proteins such as ago-2 can directly cleave messenger rna and normally repress gene expression by targeting the mrna for degradation (complete match, risc binds to mrna with perfect match) or by mediating translation inhibition or mrna deadenylation leading to mrna destabilization at the condition with mismatches between mrna sequence and the risc (incomplete match, risc binds to mrna with some mismatches). However, there are still unsettled questions regarding mirna - binding rules, thereby resulting in a lack of consensus in previous studies . Establishing direct cause - and - effect links between mirnas and mrna targets is a key to understand underlying molecular mechanisms behind health and diseases and to develop effective targeted therapeutic strategies . Now that mirnas have multiple gene targets, each target may be regulated by a suite of mirnas . The roles of mirnas in inflammation and sarcopenia have only been initially explored and future investigations will unravel their roles in immunity and metabolism . According to the analysis of mirna expression profiling, mirnas are critical regulators for both proinflammatory cytokines and skeletal muscle function [82, 83]. In order to elucidate which mirna is important in the production of proinflammatory cytokines in aging - related muscle wasting, mrna targets and specific roles in regeneration and protein synthesis in skeletal muscle need to be established . Several tissue - specific mirnas are known to be associated with the aging of skeletal muscle, which are named as myomirnas and consistently identified, including mir-1, mir-133, mir-206, mir-208, mir-486, mir-431, and mir-499 [8486]. These myomirnas can induce significant effects on development and myogenesis of skeletal muscle by targeting myogenic factors such as srf, mef2, and myostatin . Local injection of mir-206 can accelerate muscle regeneration, and mir-133 can promote the proliferation of myoblasts, while mir-1 can suppress the proliferation of myoblasts . Although there is no obvious difference in the expression of mature mir-1, mir-133, or mir-206 in skeletal muscle from younger adults, an increased expression of these primary mirnas can be observed during aging, and the effect of anabolic stimulus on the levels of these mirnas can be perturbed in the elderly . On the other hand, many studies have implicated the regulation of inflammatory response through inflammatory mirnas such as mir-155 and mir-146a, suggesting their roles in the immune system . Since inflammation is rather a broad concept, there are some overlaps between mirnas involved in inflammation and aging . Therefore, cytokine - associated mirnas appear to have central roles in both inflammation and sarcopenia . A recent rna sequencing study has demonstrated the differential expression of mirnas in skeletal muscle from old and young rhesus monkeys . Mir-181a, with its role in tuning the threshold of t - cell receptor (tcr) signaling originally described, not only acts as a myomirna, but also impacts inflammatory system . It can downregulate sirtuin 1 (sirt1) gene expression as a regulator so that the expression of mir-181a and its target gene is disrupted in aged skeletal muscle . Moreover, based on earlier reports showing proinflammatory cytokines such as tnf-, il-6, il-1, and il-8 as the proposed targets of mir-181a, the reduction of mir-181a can be responsible for an increase in the abovementioned proinflammatory cytokines in skeletal muscle during aging process . Besides, tnf- and il-1 are significantly negatively correlated with decreased expression of myomirs and can suppress the differentiation of c2c12 myoblasts to myocytes / myotubes through nf-b, jak / stat, mapk p38, or other key pathways . A newly discovered proinflammatory cytokine, tnf - like weak inducer of apoptosis (tweak), belongs to tnf family and has revealed the function for causing muscle atrophy . One of the mechanisms proposed for the induction of skeletal muscle wasting by tweak is regulating differential expression of several growth - related mirnas, including mir-1, mir-23, mir-133a, mir-133b, and mir-206 in c2c12 myotubes; however, it can reduce mir-1, mir-133a, and mir-133b only in mouse skeletal muscle . While the treatment with tweak regulates several mirnas involved in the growth of skeletal muscle, it is not known whether their regulation is a cause of muscle wasting or a compensatory response to prevent further muscle wasting . Let-7 mirna, the first known human mirna, has been reported to be critical for promoting differentiation and inhibiting cellular proliferation . The elevation of let-7 mirna may be responsible for the damage - repairing capability through the activation and proliferation of satellite cells in skeletal muscle from the elderly, therefore contributing to the attenuated regenerative capacity of skeletal muscle in the elderly . Moreover, let-7 mirna can inhibit the secretion of inflammatory cytokine il-13 in human myotubes . Recent study has also found that the overexpression of mirna let-7c can inhibit lps - induced production of tnf-, il-6, and il-1 by inhibiting the phosphorylation of stat3 . Compared to younger individuals, skeletal muscle from older individuals shows a significant elevation in let-7b and let-7e under resting conditions, suggesting the involvement of these mirnas in regulating cell cycle based on let-7 target genes . Mir-146a, which negatively regulates the expression of il-1 and il-6, is highly expressed in aged mice as a consequence of an aberrant nf-b binding to mir-146a promoter . As a result, the negative feedback regulation loop inducing the downregulation of inflammatory factors can be interrupted in aged mice . In macrophages isolated from aged mice, both dna methyl - transferase inhibitor and histone deacetylase inhibitor are able to significantly upregulate mir-146a through transcriptional activation by altering dna binding activity of nf-b . Reduced mir-146a in oxldl - activated macrophages is linked to an increase of its target, toll - like receptor 4 (tlr4), involved in lipid uptake and inflammatory cytokine secretion . Due to the upregulation in inflammatory condition in various primary disorders of skeletal muscle, mir-155 is identified as an important regulator of mef2a expression and exhibits its function in myoblast differentiation, and it also plays a critical role in the regulation of inflammation that affects both innate and adaptive immunity . The upregulation of mir-155 is the most characteristic feature of the mirna expression signature in lps - stimulated macrophages through the binding to nf-b . Moreover, the inflammatory response mediated by mir-155 is induced by tlr ligands and can enhance the translation of tnf- during the pathogenesis of metabolic syndromes . In addition, mir-23a negatively regulates pgc-1 as a key activator of mitochondrial biogenesis and function . In another study, the level of mir-696 is upregulated in skeletal muscle of mice subjected to hind limb immobilization while the level of pgc-1 as the target of mir-696 exhibits an obvious decrease . Consistent with this observation, the overexpression of mir-696 in myocytes shows a decrease in pgc-1, suggesting its involvement in mitochondrial function and metabolism, and its importance in controlling the metabolism, adaptation, and atrophy of skeletal muscle . Since a large number of mirnas have been identified in skeletal muscle, the investigation of mirnas is a promising but relatively unexplored area for understanding the regulatory mechanisms of wasting process associated with cytokine expression and secretion in aging skeletal muscle . Clearly targeting these mirnas could provide an efficient and noninvasive approach for the diagnosis, prevention, or treatment of sarcopenia through the regulation of proinflammatory and anti - inflammatory factors . Potential interventions for sarcopenia include nutritional supplements, physical activity / resistance exercise, caloric restriction, anabolic hormones, anti - inflammatory agents, and antioxidants . A key question is whether sarcopenia is a truly distinct syndrome or a milder form of a cachexia continuum . It is difficult to estimate the prevalence of sarcopenia, mostly because of practical difficulties in assessing skeletal muscle mass . Sarcopenia - associated difference in the production of proinflammatory cytokines in vivo shows a prolonged inflammation activity . There remains a lack of understanding of individual contributions of various cytokines during the aging process of skeletal muscle, because of their wide - ranging effects on cell proliferation, differentiation, migration, survival, and apoptosis . As reviewed here, mirnas play major roles in the inflammatory regulation as the pivotal regulators for modulating cell functions or the critical factors for affecting the therapeutic outcome of sarcopenia . Although the central role of inflammation in sarcopenia and proinflammatory cytokines including tnf- and il-6 as the central mediators of skeletal muscle atrophy has been documented, the roles of inflammatory mirnas in mass maintenance and functional development of skeletal muscle still need to be further explored and confirmed . Whether mirnas can be used for the diagnosis of inflammatory involvement in the aging process of skeletal muscle proinflammatory cytokine - associated mirnas such as mir-146a, mir-181, and mir-21 are frequently detected at a high level in skeletal muscle; however, their functions are still not fully clear . Therefore, further exploration of their targets, regulatory networks, and functions is highly desired . In conclusion, the discovery of mirnas with regulatory capacity of inflammatory response during aging process of skeletal muscle will open a novel avenue for the diagnosis, prevention, and therapy of sarcopenia through effectively modulating inflammatory signal pathways.

Temporary henna tattoos or pseudotattoo have become increasingly widespread among children and adolescent, as a safe and economic alternative to permanent tattoos . It is well - known that allergic skin reactions to natural henna are rare, due to its extremely low rate of sensitization . In india, north of africa, china, and egypt, it is used in weddings and religious ceremonies; in occident, it is used to dye hair and cosmetics . Paraphenylenediamine (ppd), a powerful allergen, is added to the henna tattoo mixtures (black henna tattoo) to decrease application time and intensify the color . We describe the case of a 7-year - old boy who reported erythematous papular bulls - eye shaped lesions and consolidated edema primarily in the upper and lower extremities [figure 1]. He also showed an erythematous - eczematous lesion on his leg, shaped like a dolphin [figure 2], and lesions compatible with erythema multiforme - like reaction . Erythematous papular lesions contact eczema in the tattoo area dolphin shaped patch tests were performed, and we observed a high sensitivity after 48 h and moderate after 96 h. we reported a positive reaction to ppd . Henna has been used to paint the skin for adornment and religious reasons for 9000 years and in over 60 countries . Christians, jews, muslims, hindus, and buddhists have used henna as part of their religious customs . The henna is a flowering plant native to northern africa, western and southern asia in semi - arid zones, used since antiquity to dye skin . It has a great affinity for keratinocytes, and it is used to create temporary tattoos, without it being necessary to puncture the skin . Black henna contains an ingredient in addition to pure henna to achieve its ebony color . In most cases, this added ingredient is ppd, a powerful sensitizer that should not be directly applied to the skin as it may cause mild contact dermatitis . One of the most dangerous applications of this chemical is when it is added to henna because the dye is applied while the ppd is in its oxidation process, and its potential as allergen is increased . When added to henna, the concentration of ppd is often much higher than what is approved for use in hair dyes . The cause of the sensitivity to ppd is unknown; it is believed that the mechanism involved in the pathogenesis may be a reaction mediated by type iii immune complexes and associated with type iv retarded hypersensitivity . Various topicals allergens cause erythema multiforme, including topical drugs such as corticosteroids, nonsteroidal anti - inflammatory drugs, iodine povidone, imiquimod; rubber gloves; nickel and herbicides . Three possible causes of the residual hypopigmentation have been described: a reduction in melanin synthesis, selective destruction of the melanocytes, or photoleukomelanodermitis due to pigment blocking . As henna tattoos are becoming increasingly popular, prevention requires the provision of information to consumers, especially young people and their parents . It is important for the population to be aware of this circumstance and the risk entailed by sensitization to ppd . To conclude, we believe that temporary black henna tattooing should be controlled by health authority legislation to minimize the appearance of new cases of reaction to ppd and the serious and permanent consequences we have presented . It is important for the population to be aware of the risk entailed by sensitization to ppd due to popular henna tattoos.

The romaleidae family accounts for more than 200 species of grasshoppers comprising three subfamilies: romaleinae, aucacrinae and trybliophorinae . It is the second most diverse family of the superfamily acridoidea occurring from semiarid regions to tropical rainforests (carbonell, 1977; roberts and carbonell, 1982; carbonell, 1984, 1986, 2002). The genus radacridium is dispersed across the northeast region of brazil, where it seems to be well - adapted to the severely arid conditions (carbonell, 1984). Two species are found in the state of pernambuco: radacridium mariajoseae, which is typical of the agreste region, and radacridium nordestinum, typical of the caatinga biome (carbonell, 1984, 1996). Cytogenetic studies involving representatives of romaleidae have revealed a great karyotypic conservation (mesa et al ., 1982, 2004; souza and kido, 1995; rocha et al ., 1997; pereira and souza, 2000). Most of analyzed species, including r. mariajoseae and r. nordestinum, presented karyotypes with 2n = 23 male, 24 female, an x0/xx sex chromosome system and exclusively acrocentric chromosomes . The constitutive heterochromatin (ch) in romaleidae is predominantly located in the pericentromeric regions of all chromosomes, as observed in xyleus angulatus, brasilacris gigas, chromacris nuptialis, radacridium nordestinum e phaeoparia megacephala (souza and kido, 1995; rocha et al ., analyses with dapi and cma3 in romaleids showed a predominance of gc - rich (cma3) regions . In xyleus angulatus and xestotrachelus robustus in contrast, few gc - rich ch regions were observed by loreto et al . (2005) in two chromacris species . C. nuptialis had only one bivalent (m6) with a pericentromeric cma3 block, whereas c. speciosa presented cma3 blocks in two autosomal bivalents, a proximal one in m6 and a telomeric one in l2 . A large part of the eukaryotic genome is formed by repetitive dna, including tandem sequences mainly comprising satellite dna and multigene families . Ribosomal dna and histone gene families include a variable number of copies with various genome locations (charlesworth et al . Fluorescence in situ hybridization (fish) with rdna and histone genes probes has proven useful for mapping and their location and in clarifying the genome organization of several organisms . Among invertebrates, rdna gene mapping has been used in several groups, including worms (vitturi et al ., 2002), mollusks (colomba et al ., 2002), insects (cabrero and camacho, 2008; cabral - de - mello et al ., 2011a; panzera et al ., 2012), the studies involving histone genes mapping in grasshoppers included some species of the acrididae family (cabrero et al ., 2009; oliveira et al ., 2011) and four species of the proscopiidae family (cabral - de - mello et al ., 2011b) were mapped . This study aimed at understanding the pattern of organization of multigene families in two species of radacridium . Our results showed a great variability in the number and location of rdna clusters in both species, whereas the histone h4 genes were highly conserved in number . These results are compared with data from other grasshopper species and are discussed based on the possible mechanisms involved in repetitive dna diversification . We analyzed ten individuals of r. mariajoseae from gravat (081204 s; 353353 w) and bezerros (081400 s; 354749 w) and ten adult males of radacridium nordestinum from surubim (074959 s; 354517 w), all located in the agreste region of the state of pernambuco, brazilian northeast . Specimens were processed and their testes were fixed in carnoy solution (3:1 ethanol: acetic acid). Chromosome preparations were obtained by the testicular follicles squashing technique, in one drop of 45% acetic acid . The slides were stained with cma3 (0.5 mg / ml) for one hour, washed with distilled water and stained with da (distamicine a, 0.1 mg / ml) for 45 min . The slides were rewashed and stained with dapi (2 g / ml) for 20 min and mounted in glycerol / mcilvaine buffer / mgcl2 . Probes were obtained by pcr performed according to ayres (2002), with modifications, using genomic dna from both species and the primers: 18s dnar - sca18s1f (f 5 - ccc cgt aat cgg aat gag ta - 3); sca18s1r (r 5- gag gtt tcc cgt gtt gag tc -3), 5srdna f(5- aac gac cat acc acg ctg aa -3); r (5- aag cgg tcc ccc atc taa gt - 3), histone h4 f-1 (5 - tsc gig aya aca tyc agg gia tca c - 3) and r-1 (5 - cky tti agi gcr tai acc acr tcc at - 3). The pcr products analyzed after electrophoresis in a 1% agarose gel had the expected sizes . The 18s rdna and histone h4 probes were labeled with biotin-16-dutp and the 5s rdna probe, with digoxigenin-11-dutp . The chromosome preparations were submitted to an alcohol series pretreatment and then treated with rnase and pepsin . The hybridization mix contained 1 l of probe and the denaturation was performed in a humid chamber at 75 c, followed renaturation overnight at 37 c . Immunodetection was performed with mouse anti - biotin (m743, dako) and rabbit anti - mouse - tritc (r270, dako) for biotin and sheep anti - digoxigenin (roche 1 207 741) and sheep anti - rabbit - fitc (dako - f0135) for digoxigenin . Chromosomes were counterstained with 4,6 diamidine-2-phenyl indole (dapi) and the slides were mounted with vectashield (vector). Images were obtained in a leica epifluorescence microscope, captured with the cw4000 program (leica) and adjusted in adobe photoshop cs5 . Radacridium mariajoseae and r. nordestinum presented 2n = 23(male), a sex determination mechanism of the x0(male) type and exclusively acrocentric chromosomes . Chromosomes of both species were grouped in three large (l1-l3), five medium (m4-m8) and three small (s9-s11) pairs . Cma3/da / dapi sequential staining showed gc - rich (cma3 positive) constitutive heterochromatin (ch) blocks in the interstitial region of only one medium - sized bivalent (m5) and in the pericentromeric region of the x chromosome in r. mariajoseae (figure 1a). In r. nordestinum, cma3 blocks were present in the interstitial region of the l2 bivalent and in the pericentromeric region of all chromosomes, except the l1 bivalent (figure 1c). After fish, the 18s rdna probe labeled a single site in the pericentromeric region of the x chromosome in r. mariajoseae (figure 2a) and three pericentromeric sites, in the bivalents l2, s9 and s10 of r. nordestinum (figure 2b). The 5s rdna genes were located in the pericentromeric region of the two largest bivalents (l1 and l2) and in the x chromosome of r. mariajoseae, (figure 2c) and in the bivalents l2, l3 m4 and m5 of r. nordestinumm (figure 2d). The histone h4 probe was mapped in a proximal location in a medium - sized autosome bivalent (m5) in both species (figure 2e and f). The karyotypic similarities detected after conventional analysis between the two species of radacridium did not extend to their cma3 and dapi staining patterns . Our results showed gc - richness ch heterogeneity . In r. mariajoseae only one autosome pair and the x chromosome presented cma3 blocks, which resembled the scarcity in gc - rich regions of other romaleidae, as two species of chromacris with a single cma3 block (loreto et al ., 2005). The presence of a large number of cma3 blocks is more frequent in romaleidae, as observed in xyleus angulatus, phaeoparia megacephala and xestotrachelus robustus, in which all ch was shown to be gc - rich (souza et al ., 1998; pereira and souza, 2000; souza et al ., 2003). A great difference in the 18s and 5s rdna distribution r. mariajoseae presented a single 18s rdna site in the x chromosome, confirming the finding of only one active nucleolus organizer region (nor) after silver nitrate impregnation (agno3) by rocha et al . (1997). Although three autosome bivalents showed 18s hybridization signals in r. nordestinum, only the l2 pair had a corresponding active nor identified by rocha et al . These data indicate a possible dispersion of the 18s rdna sequences, which may have been caused by structural chromosome rearrangements, ectopic recombination and transpositions (cabrero and camacho, 2008). These kind of events have been observed in other insects (nguyen et al ., 2010; cabral - de - mello et al ., the ancestral rdna site could have been present in the m9 bivalent in a common ancestor . Two points support this hypothesis: the presence of a 18s rdna site in this pair in r. nordestinum, which was considered as a megameric chromosome by rocha et al . (1997) and the preferential localization of nors is in this type of chromosome in several species of grasshoppers (rufas et al ., 1985). The 18s rdna probe location in the x chromosome of r. mariajoseae could have resulted from a chromosome rearrangement, such as a translocation or transposition, that moved it from m9 to the x, as a meiotic association between this chromosome and the megameric one has been described (loreto et al ., 2008a). 18s rdna sites restricted to autosomes, as observed in r. nordestinum, were also detected in xestotrachelus robustus, chromacris nuptialis and c. speciosa (souza et al . On the other hand, 18s rdna sites located both in autosomes and in the sex chromosome have been described, for example, in xyleus discoideus angulatus (souza et al . 18s rdna sites have been frequently observed associated with gc - rich regions in romaleidae (pereira and souza, 2000; loreto et al ., 2005), 2004), proscopiidae (souza and moura, 2000) and ommexechidae (carvalho et al ., 2011), a pattern also observed in the two species analyzed herein . The histone h4 genes were located in a single chromosome pair (m5) in both species . A single chromosome pair bearing histone genes was also described in other species of grasshoppers . (2009) analyzed the location of the histone h3 and h4 genes in 35 species of grasshoppers from the acrididae family . They observed that the great majority of species analyzed showed only one histone site, located in an autosome pair . In the same work, double fish performed in 11 randomly chosen species revealed that, in all cases, both genes were present in the same chromosome site, indicating a great conservation of histone gene location in acrididae . Cabral - de - mello et al . (2011b), using a histone h3 probe in four species of proscopiidae (tetanorhynchus silvai, scleratoscopia protopeirae, s. spinosa and stiphra robusta), observed a single site in the m4 bivalent in all the species . Oliveira et al . (2011) observed multiples sites of histone h3 in rhammatocerous brasiliensis (acrididae) and concluded that this would be a derived condition and the presence of a single histone site, as observed in both species studied herein, would be the ancestral form . The 5s and the 18s genes co - localized in the l2 chromosome of r. nordestinum and in the x chromosome of r. mariajoseae . Similarly to what we observed in both radacridium species, an extensive variation in 5s rdna distribution has been found in others grasshoppers, in which species presenting single sites and extending to all chromosome pairs have been described (loreto et al ., 2008b; cabral - de - mello et al ., 2011a). The results obtained in this study indicate a great level of karyotypic differentiation between r. mariajoseae and r. nordestinum . These data reinforce the fact that the high conservation observed at the chromosome level, including chromosome number and morphology, in radacridium and other romaleids, is not reflected at the genomic level . Our results also contribute to the understanding of chromosome evolution patterns in the family romaleidae.

The relation between chronic subclinical low - grade inflammation and insulin resistance (ir) has long been known [1, 2]. Ir is the major contributor and mediating factor in the development of type 2 dm (t2 dm) along with concomitant hypertension (ht) and cardiovascular disease (cvd) [3, 4]. The relationship between the development of dm and some markers of inflammation such as c - reactive protein (crp), il-6, fibrinogen, and pai-1 has been described previously . Serum concentration of crp increases in both impaired glucose tolerance (igt) and overt t2 dm [3, 510]. On the other hand, some studies reported that elevation of crp is an indicator of development of t2 dm . Compared with the conventional ogtt (2-hpg) as recommended by who as gold standard, fasting plasma glucose (fpg) and hba1c are more convenient, simpler, and cost - effective diagnostic methods that are currently in use for the diagnosis of t2 dm [5, 1217]. However, each test recognizes people with different metabolic features and groups who may be diagnosed by different tests but do not overlap substantially . While high postchallenge plasma glucose is a strong predictor of cvd, fasting glucose is not an independent predictor of cvd . To the best of our knowledge, there is no previous report specifically comparing the role of hs - crp in people with newly diagnosed dm with the criteria based on the 2-hpg, fpg, and hba1c . Therefore, the aim of this study was to identify the optimal cut - off points of hs - crp in new - onset (previously undiagnosed) people with dm diagnosed based on the current 2-hpg, fpg, and hba1c diagnostic criteria . In this study, hs - crp results obtained from a nationally representative population - based survey are being reported . Data derived from the turkish epidemiology survey of diabetes, hypertension, obesity and endocrine diseases (turdep - ii), a population - based study, which was included randomly assigned 26,499 adult people from 270 urban and 270 rural centers . The field survey was performed between january and june 2010, with a participation rate of 85% . The study was approved by the local ethical board (istanbul medical faculty ethical committee, 16.4.2008/699). People with known dm or other systemic diseases who had hs - crp levels of 10 mg / l (95.2 nmol / l) or above were excluded from this study due to a possible infection . All biochemical tests including glucose, insulin, and lipid profile were measured in fasting blood samples using roche diagnostics modular autoanalyzer system (roche diagnostics, germany) in the central biochemistry laboratory of istanbul medical faculty . Concentration of hs - crp was analyzed by immunoturbidimetric assay (roche / hitachi 912, modular p analyzers: acn 210; crpl3 tina - quant c - reactive protein gen . 3) and hba1c by turbidimetric inhibition immunoassay; both the system and the laboratory have been regularly certified (roche diagnostics tq hba1c gen . 3; ngsp certificate of traceability; september 2010 - 2011). A detailed medical history of each participant was obtained, and measurements of anthropometry (height, weight, waist, and hip circumference) and systolic and diastolic blood pressure (sbp, dbp) were done . Body mass index (bmi), homa - ir (= fasting glucose fasting insulin/405), and non - hdl - cholesterol (= total cholesterol hdl - cholesterol) were calculated accordingly . Glomerular filtration rate (egfr) was estimated using chronic kidney disease epidemiology collaboration ckd - epi equation . Risk factors for dm were evaluated using chi - square test; mean values by sex were compared using nonparametric mann - whitney u test . Homogeneity of variance and normal distribution of variables were tested by kolmogorov - smirnov test . Pearson correlation coefficients (r values) were calculated to assess the association between hs - crp and other laboratory parameters . Mean levels of hs - crp in each of the new dm groups were further compared using univariate analysis after being adjusted for age, bmi, smoking and alcohol drinking, and sbp and dbp . An optimum cut - off point of hs - crp was estimated using the receiver operating characteristic (roc) curve and area under the curve (auc) with 95% cis was calculated for each of the new dm groups that were diagnosed with fpg, 126 mg / dl (7.0 mmol / l), 2-hpg: 200 mg / dl (11.1 mmol / l), or hba1c: 6.5% (48 mmol / mol) [13, 17]. Optimum cut - off points of hs - crp were defined for men and women, separately . As the raw data not normally distributed when log transformed, we obtained a square root transformation (sqrt) of the hs - crp levels, and after defining the cut - off points we recalculated the squares . Data were analyzed using spss for windows (version 21.0; spss / ibm, chicago, il). Demographic characteristics and laboratory findings of women and men in turdep - ii study are presented in table 1 . In brief, men were significantly older and had higher mean weight, waist circumference, sbp, dbp, triglycerides (tg), hdl - cholesterol, and non - hdl - cholesterol values than women . Women had significantly higher bmi, hip circumference, heart rate, fpg, hba1c, 1-hpg, 2-hpg, hdl - cholesterol, and egfr values than men . Median (interquartile range, iqr) concentration of hs - crp in women was significantly higher than in men (women, 1.85 [3.09] total serum cholesterol, fasting serum insulin, and homa - ir values did not differ between men and women (table 1). There was a positive correlation between hs - crp levels and age, bmi, waist, hip, sbp, dbp, pulse, fpg, hba1c, 1-hpg, 2-hpg, tg, non - hdl - cholesterol, and homa - ir; and there was a negative correlation with hdl - cholesterol and egfr . When we repeated the analysis after controlling for ht, age, sex, smoking and alcohol use, bmi, and waist circumference, the positive correlations between hs - crp levels and hba1c, 1-hpg, 2-hpg, tg, non - hdl - cholesterol, egfr, and homa - ir and the negative correlation with hdl - cholesterol and creatinine remained significant (table 2). Among people with new dm, the highest hs - crp level was in the group detected by hba1c criterion (hs - crp median [iqr]; hba1c: 3.45 [3.82] mg / dl, 32.9 [36.4] nmol / l; 2-hpg: 2.7 [3.14] mg / dl, 25.4 [29.9] nmol / l; and fpg: 2.4 [3.0] mg / dl, 22.4 [28.5] nmol / l, data not shown). High sensitive crp level was significantly higher in women than men with newly diagnosed dm groups based on 2-hpg, fpg, and hba1c criteria . Among the newly diagnosed dm groups, the median [iqr] level of hs - crp was highest in those detected with hba1c in both genders (hba1c - group: women: 4.0 [4.1] mg / dl, 38.4 [39.1] nmol / l; men: 2.7 [3.1] mg / dl, 25.9 [29.1] nmol / l; fpg group: women: 3.3 [4.1] mg / dl, 31.1 [39.3] nmol / l; men: 2.4 [3.0] mg / dl, 22.4 [28.5] nmol / l; and 2-hpg group: women: 2.8 [3.3] mg / dl, 26.5 [31.3] nmol / l; men: 2.4 [2.8] mg / dl, 23.3 [26.9] sex differences in hs - crp did not change after the data adjusted with respect to age, bmi, waist circumference, and ht; the mean hs - crp was highest in newly diagnosed patients based on hba1c criterion in both sexes . Again, the average hs - crp levels of women were higher than men (table 3). In women with newly diagnosed dm based on fpg, mean hs - crp level was comparable to those diagnosed with 2-hpg but lower than the group diagnosed with hba1c (p = 0.000032). Women with newly detected dm based on 2-hpg had also lower mean hs - crp than those detected with hba1c (p <0.000001). In contrast, men with newly detected dm based on fpg criterion had lower mean hs - crp than those diagnosed with 2-hpg (p = 0.017) and hba1c (p = 0.003) but mean hs - crp levels were comparable in 2-hpg and hba1c based new dm groups (table 3). The specificity and sensitivity of the optimal cut - off points for hs - crp to detect dm in women were for the fpg group, 60% and 57% for 2.5 mg / l (23.6 nmol / l), 2-hpg group, 60% and 54% for 2.1 mg / l (19.7 nmol / l), and 65% and 64% for hba1c group, 2.9 mg / l (27.5 nmol / l). In men the corresponding specificity and sensitivity values were as follows: fpg group: 60% and 57% for 1.8 mg / l (16.9 nmol / l); 2-hpg group: 60% and 57% for 1.8 mg / l (16.9 nmol / l); and hba1c group: 65% and 60% for 2.0 mg / l (19.0 nmol / l) (roc curves figure 1 and table 4). The largest auc value for hs - crp to detect dm was found in women and men when using hba1c (women: 0.700; men: 0.656). The positive and negative predictive values (ppv and npv) corresponding to the above - mentioned cut - points of hs - crp in women were calculated as 58% and 59% for fpg, 57% and 58% for 2-hpg, and 64% and 65% for hba1c and in men were 58% and 59% for fpg, 58% and 59% for 2-hpg, and 61% and 61% for hba1c . Our current population - based study identified 1,727 people with newly diagnosed dm based on at least one of the three methods . However, the people identified to have dm were substantially different for each of the three methods . In other words, the concordance rate for dm among the different methods of glycaemia testing was low . Characteristics of the people with dm and their risk factors may vary by the method used for the detection of dm . Several studies have suggested that inflammation is associated with ir that takes part in the pathogenesis of t2 dm and atherosclerotic disease [14, 6]. Environmental factors such as infections, overnutrition, and lack of physical activity are believed to contribute serum crp levels although the mechanism is not properly understood . On the other hand, hyperglycaemia per se may induce inflammation and this may enhance the development of dm [21, 22]. Demonstrated that people who developed dm (detected by an ogtt) had higher baseline serum crp levels than those who did not develop dm . There was a linear increasing trend in the incidence of dm as the baseline crp quartile increased . In pizarra prospective study, people with baseline hs - crp 3 mg / l (28.6 nmol / l) developed dm . In our previous report, we demonstrated a linear increasing trend for hs - crp levels from normal glucose tolerance through impaired fasting glucose (ifg), igt, and new dm . A recent meta - analysis including 18 prospective studies demonstrated that high baseline crp levels associated with future t2 dm diagnosed based on fpg and/or 2-hpg criteria . All these findings support the chronic low - grade inflammation hypothesis in the development of dm . In our study, we found a positive correlation between hs - crp levels and all glycaemia and ir parameters . However, after adjustment for age, sex, smoking, bmi, waist, and ht, positive correlations were maintained with hba1c, 1-hpg and 2-hpg, fasting insulin, and homa - ir but not with fpg . In their later report festa et al . Stated that postchallenge glucose but not fpg was strongly correlated with baseline crp . Other studies have also shown an association between crp and dm, which remained significant after adjusting for bmi or other covariates . Our findings and others suggested that adiposity is not sufficient to explain the relationship between high levels of inflammatory markers and increased dm risk . The predictive value of hs - crp did not seem to be fully independent of obesity in several [3, 23, 24, 27, 28] but not all studies [7, 9, 10, 22]. Our results showed that among people with new dm the highest hs - crp levels were obtained in those identified with the hba1c criterion . Hba1c elevation at diagnosis is an indication of overt dm, but in a more advanced stage compared with new - onset dm detected with the fpg or 2-hpg criteria . As we did show a positive correlation between hs - crp and hba1c, it has been reported that people with dm with poorer glycaemic control had higher crp levels . Some commonly used medications like aspirin and statin may synergistically reduce serum crp concentrations [29, 30]. In the present study, we excluded people with known systemic disease and those who self - reported regularly using such medications; however, we may not ascertain all people using aspirin and/or statin . Aronson et al . Reported that crp levels among middle - aged people were higher in those with dm and ifg when compared with the healthy subjects . Similar to our findings showing women had higher hs - crp levels than men regardless of having dm or not, the adopt investigators have reported that hs - crp levels in women were higher than in men in both with and without the metabolic syndrome (ms). They reported a positive correlation between hs - crp and hba1c, bmi and homa - ir and the number of ms components in people with new dm . In women's health study, the incidence of dm was four times higher in women with hs - crp levels in the upper quartile in comparison with those with hs - crp levels in the lowest quartile . Wu and coworkers reported that high levels of hs - crp were correlated with high levels of hba1c and fpg in men and with only fpg in women . Other studies reported strong correlations between hs - crp and fasting insulin, homa - ir, and fpg [21, 34] and an inverse correlation with crp and hdl - cholesterol [3, 21]; all were confirmed in our study . We estimated the optimal cut - off hs - crp and auc - crp with 95% cis for dm for each of the three diagnostic methods separately . To the best of our knowledge, this is the first study aiming at determining hs - crp cut - off points indicating new dm compared with each of the three methods assessing hyperglycaemia . The highest cut - off point for hs - crp was obtained with hba1c based new dm detection compared to fpg and 2-hpg methods . In fact we previously showed that new dm group detected with hba1c has a more advanced metabolic disorder (higher bmi, waist, blood pressure, non - hdl - cholesterol, triglycerides, and insulin but lower hdl - cholesterol) than other new dm groups detected with fpg or 2-hpg . Den engelsen et al . Attempted to find a cut - off point for hs - crp that would indicate the presence of the ms . If hs - crp cut - off point was set at 3 mg / l (28.6 nmol / l), the sensitivity and specificity were 72% and 37%; at this point ppv and npv were 42% and 67% . In another study performed in a japanese population, a cut - off point of hs - crp of 0.65 mg / l (6.2 nmol / l) for fpg 100 mg / dl (5.6 mmol / l) or higher was capable of defining the ms with a 100% sensitivity and 77% specificity in women and with a 65% sensitivity and 63% specificity in men . In our study the optimum cut - point of hs - crp of 2.5 mg / l (23.6 nmol / l) in women and 1.8 mg / l (16.9 nmol / l) in men for fpg 126 mg / dl (7.0 mmol / l) or higher was capable of defining the new dm with a 60% sensitivity and 57% specificity in both genders . The optimum cut - point for hba1c 6.5% and over was 2.9 mg / l (27.5 nmol / l) in women with a 65% sensitivity and 64% specificity and 2.0 mg / l (19.0 nmol / l) in men with a 60% sensitivity and 62% specificity, and the largest auc value for hs - crp to detect new dm was found in women and men when using hba1c (women: 0.700; men: 0.656). These high cut - off points of crp may be related with the more advanced diabetic state as compared to fpg or 2-hpg based detected cases . One of the greatest strengths of the present study is its national representative sampling with a large sample size and wide age range . In addition, this is the first study where all three currently proposed methods (2-hpg, fpg, and hba1c) were used to define dm and each of them was compared with the inflammation marker, hs - crp . The major limitations are the cross - sectional design and somewhat higher participation rate in women, controlled in the data analyses . In brief, an hs - crp level 1.8 mg / l (16.9 nmol / l) generally detects more than half of the people with new dm . External validation of our findings needs to be carried out in additional studies in other populations with reasonably large sample size before these findings can be generalized . Our results revealed that hs - crp may not further strengthen the diagnosis of new - onset dm . However, the highest hs - crp among people with new dm was found in those identified with the hba1c criterion . This suggests that high hba1c may recognize new dm cases at a more advanced stage than fpg or 2-hpg in an ogtt . . It would be important to find out if people with newly detected dm with high hs - crp require a more intensive therapy than those with low hs - crp.

. They can be caused by mutations in nuclear genes either relating to mitochondrial components or mitochondrial dna (mtdna). The prevalence of mitochondrial diseases due to mutations in mtdna and related nuclear genes is estimated to be 1 in 5000 . Mutations in mtdna are much higher than those in the related nuclear genes, probably because that mitochondrial genome has been exposed in reactive oxygen species(ros) and lacked protection structure . In 1990, the adenine to guanine transition at the mtdna position of 3243 encoding trna was found to be the molecular basis for mitochondrial encephalomyopathy with lactic acidosis and stroke - like episodes (melas). Epidemiological studies showed that m.3243a> g mutation is the most frequent pathogenic mutation in mtdna . The prevalence of m.3243a> g mutation in mtdna is 3.5/100,000 adults in the north east of england and approximately 1/6000 in an adult population in finland . Seizures, encephalopathy, and stroke - like episodes were found in about 80% patients with melas, and short stature, deafness, cognitive impairment, exercise intolerance, migraines, depression, cardiomyopathy, cardiac conduction defects, and diabetes mellitus were also found in some melas cases . Here, we summarized the clinical spectrum of m.3243a> g mutation in chinese pediatric patients, to define the common clinical manifestations and study the correlation between heteroplasmic degree of the mutation and clinical severity of the disease . Clinical data of 100 chinese pediatric patients who were first diagnosed as mitochondrial diseases through gene test for m.3243a> g mutation in peking university first hospital from 2007 to 2013 were retrospectively reviewed . This study was approved by the medical ethics committee of peking university first hospital (no . A total of ten clinical characteristics including vision impairment, hearing loss, encephalopathy, myopathy, and gastrointestinal disturbances were collected from these patients . Short stature was defined as the body height less than two standard deviations below the mean height of normal children . The peripheral blood samples were collected from these patients when they were first screened for the gene mutation . Detection of m.3243a> g mutation ratio was performed by polymerase chain reaction (pcr)-restriction fragment length polymorphism method . The pcr product was digested with restriction enzyme apa i, and then separated in 2% agarose gel . The patients were divided into three groups based on mutation ratio (low level: 030%; middle level: 3160%; and high level: 61100%). Age and mutation ratio inconsistent with gaussian distribution were presented as median (q1, q3). Correlation between m.3243a> g mutation ratio and age was evaluated with spearman's rank correlation method . The differences in clinical symptom frequency of patients with low, middle, and high levels of mutation ratio were analyzed by chi - square test . Clinical data of 100 chinese pediatric patients who were first diagnosed as mitochondrial diseases through gene test for m.3243a> g mutation in peking university first hospital from 2007 to 2013 were retrospectively reviewed . This study was approved by the medical ethics committee of peking university first hospital (no . A total of ten clinical characteristics including vision impairment, hearing loss, encephalopathy, myopathy, and gastrointestinal disturbances were collected from these patients . Short stature was defined as the body height less than two standard deviations below the mean height of normal children . The peripheral blood samples were collected from these patients when they were first screened for the gene mutation . Detection of m.3243a> g mutation ratio was performed by polymerase chain reaction (pcr)-restriction fragment length polymorphism method . The pcr product was digested with restriction enzyme apa i, and then separated in 2% agarose gel . The patients were divided into three groups based on mutation ratio (low level: 030%; middle level: 3160%; and high level: 61100%). Age and mutation ratio inconsistent with gaussian distribution were presented as median (q1, q3). Correlation between m.3243a> g mutation ratio and age was evaluated with spearman's rank correlation method . The differences in clinical symptom frequency of patients with low, middle, and high levels of mutation ratio were analyzed by chi - square test . The age of the diagnosis of mitochondrial disease ranged from 2 months to 18 years, with the median age of 9 years (5.8 years, 12.0 years). The m.3243a> g mutation ratio varied from 5% to 93%, with a median level of 44% (36%, 54%). There was no significant difference in m.3243a> g mutation ratio between males and females (t = 0.691, p = 0.491). Patients at the onset had one or more symptoms, including seizures (54%), muscle weakness (29%), headache complicated with vomiting (25%), decreased vision (18%), hearing loss (10%), impaired verbal communication (6%), and heart preexcitation syndrome (5%). The most prevalent symptom of these patients was seizures (76%), followed by short stature (73%), elevated plasma lactate (70%), abnormal magnetic resonance imaging / computed tomography (mri / ct) changes (68%), vomiting (55%), decreased vision (52%), headache (50%), and muscle weakness (48%). Most of the patients were multisymptomatic, only two patients had one symptom, and five patients manifested two symptoms . Seizures were present in 76 patients (76%), of which 13 patients had stroke during or shortly after seizures . Recurrent headaches were found in 50 patients (50%), of which most were complicated with vomiting and/or vision loss . Sixty - eight patients (68%) were found to have mri / ct abnormalities, including abnormal asymmetric signals in occipital area (51/68, 75%), temporal area (21/68, 31%), parietal area (20/68, 29%), bilateral basal ganglia calcification (16/68, 24%), cerebral atrophy (10/68, 15%), and thalamus and brainstem lesions (5/68, 7%). Slurred speech was present in 16 patients, of whom 3 had progressively worsening speech, and 2 had delayed speech development (at 1.5 and 3.0 years of age). Seventy - three patients (73%) had a short stature and 69 patients (69%) had a weight loss . Fifty - five patients (55%) had experienced recurrent vomiting and 38 patients (38%) had diarrhea or / and constipation . Plasma lactate ranged from 1.4 to 19.0 mmol / l (normal range: 0.72.0 mmol / l). Elevated plasma lactate was detected in 70 patients (70%), of which 90% had a plasma lactate> 5 mmol / l . Vision impairment was found in 52 patients (52%), of which 33 experienced blurred vision and 16 had decreased visual acuity . Reduced muscle strength was reported in 48 patients (48%), of which 31 had minimal muscle weakness in upper limbs and shoulders and 17 manifested muscle weakness in lower limbs, and 2 had walk difficulties . Thirty - seven patients (37%) complained of difficulties in maintaining stability, of them 18 experienced frequent tumbling, and 38 patients (38%) had compromised exercise tolerance, which appeared during running or going upstairs in most cases and during walk on flat places in only two cases . Heart diseases were detected in 35 patients (35%), of which 17 were found to have abnormal electrocardiogram such as st - t changes and arrhythmias . Left ventricular hypertrophy was found in eight patients, right ventricular hypertrophy in five patients, and preexcitation syndrome in five patients . Twenty - one patients (21%) had hearing impairment, presenting tinnitus or hearing loss (mild deafness in 14 patients, moderate deafness in 5 patients, and severe deafness in 2 patients). The m.3243a> g mutation ratio in peripheral leukocytes was determined in all the patients, and 32% of them had a mutation ratio above 50% . The relationship between m.3243a> g mutation ratio and onset age was analyzed by spearman's rank correlation method, which showed that m.3243a> g mutation ratio was correlated negatively with onset age [r = 0.470, p <0.001; figure 1]. Correlation between m.3243a> g mutation ratio in peripheral leukocytes and onset age (r=0.470, n=100). The differences in clinical symptom frequency among patients with low, middle, and high levels of mutation ratio were analyzed by chi - square test [table 1]. There were significant differences in the frequencies of hearing loss, decreased vision, myopathy, and gastrointestinal disturbance among three groups . However, patients with a low or middle level of m.3243a> g mutation ratio were more likely to suffer from hearing loss, decreased vision, and gastrointestinal disturbance than high level group . Analysis of clinical symptom frequency in different distributions of 3243a> g mutation ratio, n low level: a mutation ratio range 030%; middle level: a mutation ratio range 3160%; high level: a mutation ratio range 61100%; mri: magnetic resonance imaging; ct: computed tomography . Patients at the onset had one or more symptoms, including seizures (54%), muscle weakness (29%), headache complicated with vomiting (25%), decreased vision (18%), hearing loss (10%), impaired verbal communication (6%), and heart preexcitation syndrome (5%). The most prevalent symptom of these patients was seizures (76%), followed by short stature (73%), elevated plasma lactate (70%), abnormal magnetic resonance imaging / computed tomography (mri / ct) changes (68%), vomiting (55%), decreased vision (52%), headache (50%), and muscle weakness (48%). Most of the patients were multisymptomatic, only two patients had one symptom, and five patients manifested two symptoms . Seizures were present in 76 patients (76%), of which 13 patients had stroke during or shortly after seizures . Recurrent headaches were found in 50 patients (50%), of which most were complicated with vomiting and/or vision loss . Sixty - eight patients (68%) were found to have mri / ct abnormalities, including abnormal asymmetric signals in occipital area (51/68, 75%), temporal area (21/68, 31%), parietal area (20/68, 29%), bilateral basal ganglia calcification (16/68, 24%), cerebral atrophy (10/68, 15%), and thalamus and brainstem lesions (5/68, 7%). Slurred speech was present in 16 patients, of whom 3 had progressively worsening speech, and 2 had delayed speech development (at 1.5 and 3.0 years of age). Seventy - three patients (73%) had a short stature and 69 patients (69%) had a weight loss . Fifty - five patients (55%) had experienced recurrent vomiting and 38 patients (38%) had diarrhea or / and constipation . Plasma lactate ranged from 1.4 to 19.0 mmol / l (normal range: 0.72.0 mmol / l). Elevated plasma lactate was detected in 70 patients (70%), of which 90% had a plasma lactate> 5 mmol / l . Vision impairment was found in 52 patients (52%), of which 33 experienced blurred vision and 16 had decreased visual acuity . Reduced muscle strength was reported in 48 patients (48%), of which 31 had minimal muscle weakness in upper limbs and shoulders and 17 manifested muscle weakness in lower limbs, and 2 had walk difficulties . Thirty - seven patients (37%) complained of difficulties in maintaining stability, of them 18 experienced frequent tumbling, and 38 patients (38%) had compromised exercise tolerance, which appeared during running or going upstairs in most cases and during walk on flat places in only two cases . Heart diseases were detected in 35 patients (35%), of which 17 were found to have abnormal electrocardiogram such as st - t changes and arrhythmias . Left ventricular hypertrophy was found in eight patients, right ventricular hypertrophy in five patients, and preexcitation syndrome in five patients . Twenty - one patients (21%) had hearing impairment, presenting tinnitus or hearing loss (mild deafness in 14 patients, moderate deafness in 5 patients, and severe deafness in 2 patients). The m.3243a> g mutation ratio in peripheral leukocytes was determined in all the patients, and 32% of them had a mutation ratio above 50% . The relationship between m.3243a> g mutation ratio and onset age was analyzed by spearman's rank correlation method, which showed that m.3243a> g mutation ratio was correlated negatively with onset age [r = 0.470, p <0.001; figure 1]. Correlation between m.3243a> g mutation ratio in peripheral leukocytes and onset age (r=0.470, n=100). The differences in clinical symptom frequency among patients with low, middle, and high levels of mutation ratio were analyzed by chi - square test [table 1]. There were significant differences in the frequencies of hearing loss, decreased vision, myopathy, and gastrointestinal disturbance among three groups . However, patients with a low or middle level of m.3243a> g mutation ratio were more likely to suffer from hearing loss, decreased vision, and gastrointestinal disturbance than high level group . Analysis of clinical symptom frequency in different distributions of 3243a> g mutation ratio, n low level: a mutation ratio range 030%; middle level: a mutation ratio range 3160%; high level: a mutation ratio range 61100%; mri: magnetic resonance imaging; ct: computed tomography . The m.3243a> g mutation has been shown to lead to reduced levels of the trna, decrease in aminoacylation, and absence of the normal modification with 5-taurinomethyl group at the wobble base . Mitochondrial disease caused by this mutation may result from the reduction of mtdna - encoded proteins and oxidative phosphorylation activity in mitochondrial translation . Several clinical syndromes including melas, myoclonic epilepsy with ragged - red fibers, chronic progressive external ophthalmoplegia, and leigh's syndrome may associate with m.3243a> g mutation . Patients with m.3243a> g mutation but without clinical symptoms are not uncommon . To define the spectrum of clinical manifestations due to m.3243a> g mutation, we retrospectively reviewed clinical data of 100 chinese pediatric patients with m.3243a> g mutation regardless of their clinical presentations . Central nervous system (cns) is frequently involved in mitochondrial diseases because of its higher energy demand . Seizures may result from neuronal energy depletion, oxidative stress, impaired calcium signaling, immune disturbances, and deficiencies of vitamins, cofactors, and amino acids . In this study, the prevalence of seizures was as high as 76%, and was nearly 100% in our previous study, similar to the prevalence of 70.5% in a report of pediatric mitochondrial disease from taiwan, china . Cns symptoms are, therefore, more common and severe in children than adults . The prevalence of seizures ranged from 9% to 20% in adults . In melas patients, recurrent stroke - like episodes were more frequent in those with m.3243a> g mutation than those without the mutation . We found stroke - like episodes in 22% patients, higher than the prevalence from patients with mitochondrial mutations other than m.3243a> g mutation . Most of our patients showed abnormal brain image findings were probably not due to vascular injuries . Bilateral basal ganglia were the most vulnerable site in this disease, followed by temporal, parietal, and occipital area, similar to the previous report . Chae et al . Also observed the tendency that basal ganglia were frequently involved in patients without m.3243a> g mutation . Vomiting was more frequent than constipation or / and diarrhea, and 68% vomiting cases were complicated with headaches . A higher prevalence (42%) of these symptoms therefore, mitochondrial diseases should be considered clinically in children with unexplained vomiting and headaches . The prevalence of cardiac involvement was highly variable in patients with mtdna mutations, ranging from 17% to 40% . In this study, 35% patients exhibited cardiac involvement with the main manifestations of abnormal electrocardiogram (17%) and left ventricular hypertrophy (8%). The prevalence of left ventricular hypertrophy was less in our patients than the patients with m.3243a> g mutation (56%) reported by majamaa - voltti et al . Pediatric patients may be the reason of that lower prevalence of cardiac involvement was present in this study . For patients suspected of mitochondrial disease, routine electrocardiogram, and ultrasonocardiography the m.3243a> g mutation ratio in peripheral leukocytes was negatively correlated with patients onset age . Myopathy was frequently seen in patients with high level of m.3243a> g mutation ratio . However, patients with a low or middle level of m.3243a> g mutation ratio were more likely to suffer from hearing loss, decreased vision, and gastrointestinal disturbance . In conclusion, this study summarized the common symptoms and onset symptoms in a large cohort of chinese pediatric patients with m.3243a> g mutation . In view of the fact that the diagnosis of 66% of patients was delayed an average of 2 years, we suggested that examination of m.3243a> g mutation in mtdna should be considered in children with one or more of the symptoms including seizures, short stature, muscle weakness, headache complicated with vomiting, decreased vision, and hearing loss . Although the mutation ratio in blood sample is an available test for diagnosis of mitochondrial disease, the m.3243a> g mutation ratio is usually higher in muscle and urine sample . Therefore, the mutation ratio in other tissues, such as muscle and urine cell, should be included in the future study . This study was supported by grants from national natural science foundation of china (no . This study was supported by grants from national natural science foundation of china (no . 81271256 and no.

Prolonged exposure to microgravity has significant effects on physiological conditions . Many studies have investigated the effects of microgravity on the neuromuscular system . Most of these studies have focused on skeletal muscle changes under microgravity . However, to the best of our knowledge, there is limited research regarding the effects of microgravity on the peripheral nervous system . One study examined the conduction velocity (cv) in branching axon terminals following a space mission or bed rest and identified a decreased cv; however, it did not measure the velocity in the main trunk of nerve fibers . For example, one study investigated the manifestation of somatosensory - evoked potentials under hypokinesia, which included both the peripheral and central sensory nervous systems . However, the conditions under hypokinesia / bed rest are different compared with microgravity; therefore, the effect of microgravity on the peripheral nervous system should be further evaluated . Ground - based models are a suitable alternative to space flights and induce similar modifications in the neuromuscular system . The hindlimb unloading (hu) model is characterized by the absence of weightbearing and reduced motor activity, and it is typically applied in rats . Moreover, the 6 head - down tilt (hdt) is another experimental model used to simulate the space flight environment of humans . To determine changes in nerve conduction under simulated microgravity, the current study aimed to investigate the nerve conduction characteristics of rhesus monkeys before and after prolonged exposure to hdt . All experimental procedures conducted with rhesus monkeys were approved by the ethical committee of the china astronaut research and training center, which is in accordance with the principles of the association for assessment and accreditation of laboratory animal care international (aaalac), approved by institutional animal care and use committee (iacuc). The hdt model in rhesus monkeys was selected as the model because the peripheral nervous system of primates is the most similar to humans compared with other species . Six rhesus monkeys at the age of 34 years (three males and three females) were laid on beds, which were tilted backward 6 from the horizontal . The head - down animals wore a special cloth, which enabled them to be fixed to the bed; however, their arms and legs were free to move, and they were provided access to food and water . The hdt model functions to reduce mechanical loading to the hindlimbs, which provides a similar condition as microgravity . The rhesus monkeys were provided with a regular dietary program, which included primate biscuits, fresh fruits, and vitamin syrup . The animals were housed one per cage or bed in rooms with air temperature maintained at 23 2c and a standard 12:12 h dark light cycle (lights were turned on at 8:00 a.m. and off at 8:00 p.m.). The general health condition of the animals was carefully monitored . The monkeys were provided with toys (for example, the drum - shaped rattle, a chinese traditional toy) during housing throughout the duration of the study, and the toys were available all the time except during experimental procedures . Nerve conduction studies (ncss) were performed using surface electrodes on the median, ulnar, tibial, and fibular nerves on the right side to assess motor function in the upper and lower limbs using the key point electromyography system (31a06, medoc ltd ., the motor conduction velocities, the proximal and distal amplitudes of the compound muscle action potentials (cmaps), and the proximal and distal latencies of the median, ulnar, tibial, and fibular nerves were recorded (representative images of the ncs of the tibial nerve are shown in figure 1). The ncs was performed in the order of the median, ulnar, tibial, and fibular nerves before hdt and on 7, 21, and 42 days of hdt . During the procedure, toys were provided, and the researchers accompanied the monkeys to reduce pain and suffering . All animal housing and experiments occurred in the animal facility at china astronaut research and training center . Ncs: nerve conduction studies; hdt: head - down tilt . Statistical analysis was performed using spss software version 19.0 (ibm, chicago, il, usa). Analysis of variance (anova) using a randomized block design was conducted to compare the differences in the ncs before hdt and at 7, 21, and 42 days of hdt . Post hoc analyses, including least significant difference and student newman keuls, were performed if significant differences were identified in the anova . One rhesus monkey died on the day 21 of hdt because of inadaptation to the simulated microgravity, and one monkey injured its upper limbs as a result of rigorous movement . Thus, the anova with a randomized block design and the anova of the median and ulnar nerve parameters were performed using data collected from the remaining four rhesus monkeys . Data from the tibial and fibular nerves were obtained from the five remaining rhesus monkeys . All experimental procedures conducted with rhesus monkeys were approved by the ethical committee of the china astronaut research and training center, which is in accordance with the principles of the association for assessment and accreditation of laboratory animal care international (aaalac), approved by institutional animal care and use committee (iacuc). The hdt model in rhesus monkeys was selected as the model because the peripheral nervous system of primates is the most similar to humans compared with other species . Six rhesus monkeys at the age of 34 years (three males and three females) were laid on beds, which were tilted backward 6 from the horizontal . The head - down animals wore a special cloth, which enabled them to be fixed to the bed; however, their arms and legs were free to move, and they were provided access to food and water . The hdt model functions to reduce mechanical loading to the hindlimbs, which provides a similar condition as microgravity . The rhesus monkeys were provided with a regular dietary program, which included primate biscuits, fresh fruits, and vitamin syrup . The animals were housed one per cage or bed in rooms with air temperature maintained at 23 2c and a standard 12:12 h dark light cycle (lights were turned on at 8:00 a.m. and off at 8:00 p.m.). The monkeys were provided with toys (for example, the drum - shaped rattle, a chinese traditional toy) during housing throughout the duration of the study, and the toys were available all the time except during experimental procedures . Nerve conduction studies (ncss) were performed using surface electrodes on the median, ulnar, tibial, and fibular nerves on the right side to assess motor function in the upper and lower limbs using the key point electromyography system (31a06, medoc ltd ., the motor conduction velocities, the proximal and distal amplitudes of the compound muscle action potentials (cmaps), and the proximal and distal latencies of the median, ulnar, tibial, and fibular nerves were recorded (representative images of the ncs of the tibial nerve are shown in figure 1). The ncs was performed in the order of the median, ulnar, tibial, and fibular nerves before hdt and on 7, 21, and 42 days of hdt . During the procedure, toys were provided, and the researchers accompanied the monkeys to reduce pain and suffering . All animal housing and experiments occurred in the animal facility at china astronaut research and training center . Ncs of tibial nerve before hdt and 42 days after hdt in rhesus monkeys . (a) before hdt . Statistical analysis was performed using spss software version 19.0 (ibm, chicago, il, usa). Analysis of variance (anova) using a randomized block design was conducted to compare the differences in the ncs before hdt and at 7, 21, and 42 days of hdt . Post hoc analyses, including least significant difference and student newman keuls, were performed if significant differences were identified in the anova . One rhesus monkey died on the day 21 of hdt because of inadaptation to the simulated microgravity, and one monkey injured its upper limbs as a result of rigorous movement . Thus, the anova with a randomized block design and the anova of the median and ulnar nerve parameters were performed using data collected from the remaining four rhesus monkeys . Data from the tibial and fibular nerves were obtained from the five remaining rhesus monkeys . The parameters of ncs before hdt (under normal conditions) and 7, 21, and 42 days of hdt are presented in table 1 . The proximal amplitude of the cmap of the median nerve was significantly decreased at 21 and 42 days of hdt compared with the amplitude before hdt (4.38 2.83 vs. 8.40 2.66 mv, f = 4.85, p = 0.013 and 3.30 2.70 vs. 8.40 2.66 mv, f = 5.93, p = 0.004, respectively). The proximal amplitude of the cmap of the median nerve at 42 days of hdt was significantly decreased compared with that at 7 days of hdt (3.30 2.70 vs. 7.70 1.50 mv, f = 4.40, p = 0.022). Ncs before and at 7, 21, and 42 days of hdt in rhesus monkeys (n = 6) data represent means sds . * for the median nerve, statistically different versus the proximal cmap before hdt; for the median nerve, statistically different versus distal cmap before hdt; for the tibial nerve, statistically different versus the proximal cmap before hdt; for the median nerve, statistically different versus the proximal cmap 7 days of hdt; for the median nerves, statistically different versus distal cmap 7 days of hdt; for the tibial nerve, statistically different versus the proximal cmap 7 days of hdt . Ncs: nerve conduction study; hdt: head - down tilt; mcv: motor conduction velocity; cmap: compound muscle action potential; sds: standard deviations . The distal amplitude of the cmap of the median nerve was significantly decreased at 7, 21, and 42 days of hdt compared with the amplitude before hdt (7.28 1.27 vs. 10.25 3.40 mv, f = 4.03, p = 0.039; 5.05 2.01 vs. 10.25 3.40 mv, f = 6.25, p = 0.04; and 3.95 2.79 vs. 10.25 3.40 mv, f = 7.35, p = 0.01, respectively). The distal amplitude of the cmap of the median nerve at 42 days of hdt was significantly decreased compared with that at 7 days of hdt (3.95 2.79 vs. 7.28 1.27 mv, f = 3.33, p = 0.08). The proximal amplitude of the cmap of the tibial nerve was significantly decreased at 42 days of hdt compared with the amplitude before hdt (6.14 1.94 vs. 11.87 3.19 mv, f = 5.02, p = 0.039). The proximal amplitude of the cmap of the tibial nerve at 42 days of hdt was significantly decreased compared with that at 7 days of hdt (6.14 1.94 vs. 12.98 4.99 mv, f = 6.84, p = 0.008). The proximal and distal latencies of the nerves and the nerve cvs were prolonged and decreased slightly compared with the parameters before hdt but were not significantly different . Previous studies regarding the effects of microgravity on the neuromuscular system have mainly focused on muscle atrophy induced by muscle disuse . Few studies have investigated the electrophysiological characteristics of the peripheral nervous system after prolonged microgravity . The current investigation focused on the electrophysiological characteristics of the peripheral nervous system and identified a significant decrease in the proximal and distal amplitudes of the cmap of the median and tibial nerves as early as day 7 of hdt . Previous research identified a longer duration and a slower falling rate of the action potentials of muscle fibers, thereby reflecting a change in the motor fiber impulses of its nerve, which may contribute to the decreased amplitude . Furthermore, atypical combinations of myosin heavy chain mrna isoforms have been demonstrated to be transiently increased in the soleus fibers of rats that experienced 7 days of hu, which suggests a disruption of transcriptional and translational activity . These changes were present at a very early stage and may explain the cmap amplitude decrease in the ncs identified in the current study . Furthermore, as the recording surface electrodes were attached at the muscle belly, a decrease in muscle power and muscle atrophy may contribute to the cmap amplitude decrease of the median and tibial nerves . However, there were no significant differences in the proximal and distal amplitudes of the cmap of the ulnar and fibular nerves at 42 days of hdt . The reason for this finding may be that 42 days of hdt is not sufficient to produce dramatic differences . Studies of rats under microgravity have typically lasted for 14 days or less, and significant changes in multiple parameters such as the nerve cv and axon diameter have been identified . In humans, primates most closely resemble humans; however, 42 days of hdt was likely not sufficient to induce similar changes . Moreover, one study demonstrated that the equilibrium state in rats shifted to a different level at 14 days of hu . The transitional period between these two equilibrium states may have been marked by a transitory disorganization . We infer that the electrophysiological characteristics of peripheral nerves were under a transitory disorganization on day 42 of hdt . Thus, the amplitudes of the cmap of the ulnar and peroneal nerves did not exhibit significant decreases . . A significant decrease in the cv of muscle fibers and a reduction in the cv in branching axon terminals were previously identified . In this study, the latencies of the nerves and the nerve cvs were not significantly different after hdt . The mechanisms that modulate the cv in nerve fibers are not clear, and changes in the membrane properties at the nodes of ranvier may represent one factor that plays a role in this modulation . Changes in the membrane properties may be induced through modifications in the synthesis of voltage - gated membrane channels in excitable muscle membranes . Wittwer et al . Demonstrated gene expression alterations following prolonged unloading of rat soleus muscles . There was an increase in the mrnas that coded for ion channels, which may elucidate the increased shortening velocity of the soleus muscle following spaceflight . In our study, the most likely explanations for the lack of change in the latencies of the nerves and nerve cvs following hdt were that the 42 days of exposure to hdt was not sufficient to generate significant changes, and the electrophysiological characteristics of the peripheral nerve fibers were under a transitory disorganization . Furthermore, to the best of our knowledge, the nerve cv depends on the axonal diameter, myelin sheath thickness, and internodal distance . Previous research indicates that although myelin was thinner following hu, the mean axonal diameter for the small fibers was slightly decreased in hu rats; in contrast, it was not significantly different for the large fibers, which were in charge of motor function, and the distances between the nodes of ranvier and the global structure of the nerve fibers were not significantly different . Moreover, the current findings are consistent with previous research regarding fatigue, in which the cv along nerve fibers was not substantially reduced . In this study, rhesus monkeys were subjected to hdt to resemble the space flight environment, which is a model of substantial value in the investigation of microgravity effects on astronauts . Experiments that involve astronauts, including ground - based experiments, are expensive, and the impact of microgravity on the motor function of astronauts is often minimized through countermeasures . Furthermore, the pretest conditions of astronauts are challenging to control; thus, the potential heterogeneity may impact the reliability of the findings . Moreover, although studies have evaluated changes in muscle fibers and axon terminals under microgravity, few studies have investigated these variables in nerve trunks . The current findings provide novel evidence related to the electrophysiological characteristics of nerve trunks under microgravity . Despite the importance of the experimental model and findings identified in this study, thus, future studies with a larger sample size and a longer duration of hdt are necessary to confirm these findings . Conclusively, this study demonstrates that the cmap amplitudes of nerves are decreased under hdt in rhesus monkeys . Moreover, results of the present study suggest that rhesus monkeys exposed to hdt might be used as an experimental model for the study of ncs under microgravity.

A 55 year - old man visited our emergency department because of increasing frequency of chest pain . He had undergone off - pump coronary artery bypass grafting (cabg) 10 years ago because of unstable angina associated with three vessel coronary artery disease . At the initial operation, the in situ right internal thoracic artery (ita), in situ left ita and in situ right gastroepiploic artery (rgea) grafts were used to revascularize the left anterior descending coronary artery, two obtuse marginal coronary branches, and posterior descending coronary artery, respectively . An excess segment of the distal right ita was connected to the side of left ita as a y - composite graft and anastomosed to the first diagonal coronary artery . Coronary angiography and myocardial single photon emission computed tomography (spect) were performed at 5 years after surgery as a follow - up study . The 5-year angiography showed all patent grafts and the myocardial spect demonstrated no perfusion decrease . Exertional chest pain recurred at 7 years after surgery, and a repeated coronary angiography showed patent previous grafts including faint visualization of the in situ rgea graft associated with significant stenosis at the os of the celiac axis . The computed tomographic angiogram also demonstrated a 90% stenosis at the celiac os, which had been without stenosis on abdominal angiography taken before the surgery (fig . Redo off - pump cabg was performed 10 years after the initial surgery because of an increasing frequency of angina and an aggravated finding of the follow - up myocardial spect, which was a newly developed reversible perfusion decrease in the inferior wall (fig ., the great saphenous vein was harvested from the lower leg and interposed between the middle part of in situ right ita and distal part of in situ rgea grafts used previously, to supply blood flow from the right ita graft to the posterior descending coronary artery . One year after redo surgery, the patient had no symptoms of angina and coronary angiogram was performed and revealed patent grafts, including an interposed saphenous vein graft (fig . 3a). The myocardial spect test was also performed and demonstrated that there was no perfusion decrease including the inferior wall (fig . Reoperations for coronary artery disease have been increased due to the increased number of isolated cabg . The society of thoracic surgeons statistics indicated that nearly 5% of the current cabg procedures done in the us were repeat surgical revascularization . Angiographic indications for reoperation are progression of native coronary atherosclerosis, previous graft failure or a combination of both . One previous study demonstrated that 4 out of 400 patients who underwent cabg using the rgea graft needed percutaneous interventions due to the rgea graft failure during postoperative follow - up of 2211 months . One of those 4 patients required an angioplasty for a newly developed stenosis of the celiac trunk . In the present case, an indication for reoperation the patient had been free of angina, and the angiographic and myocardial spect follow - up studies revealed no abnormal findings at postoperative 5 years . When the patient suffered from recurred angina at postoperative 7 years, coronary angiography showed a faint visualization of the in situ rgea graft associated with significant stenosis at the os of the celiac axis . The 10-year follow - up myocardial spect test demonstrated a newly developed reversible perfusion decrease in the inferior wall . The prevalence of celiac axis stenosis was 7.3% in a korean population although it was lower than the previously reported incidence of celiac axis stenosis in western populations ranged from 12.5% to 24% . In the present case, celiac artery stenting could be an alternative option in such a case . However, we performed a redo operation because celiac axis stenting was associated with a high incidence of late restenosis . The aorta or another in situ arterial graft could be chosen as a blood source . Alternatively, patent in situ grafts used previously may be re - used as an inflow conduit . With regards to our patient, the 3 in situ arterial grafts had already been used . The saphenous vein graft was interposed between the middle part of right ita and distal part of in situ rgea grafts used previously.

To report a case of inadvertent anterior chamber and cornea stromal injection with high dose antibiotics and steroids during cataract operation . During cataract operation on a 78 year - old female patient, high dose gentamicin (20 mg/0.5 ml) and dexamethasone (2 mg/0.5 ml) were inadvertently injected into the anterior chamber and cornea stroma when making cornea edema for sealing of the incision sites . On postoperative day one, extensive cornea edema was noted, and best - corrected visual acuity was 0.2 . Postoperatively, corneal edema fully resolved, and best - corrected visual acuity was 0.8 . However, some descemet's membrane folds still remained, and a decrease in the number of endothelial cells was noted by specular microscope . In this case involving anterior chamber and cornea stromal injection with high dose antibiotics and steroids, immediate anterior chamber irrigation with balanced salt solution seemed an appropriate management, and the patient's long - term visual acuity appears good . To prevent such mistakes, precise labeling of all solutions and use of different syringe needles should be considered . A 78 year - old woman underwent extracapsular cataract extraction with phacoemulsification via temporal clear corneal and a single superior side port incision . An intraocular lens was inserted in the bag, and viscoelastic materials were removed by irrigation and aspiration, without any complications . At the end of the operation, the nurse inadvertently handed over the wrong syringe and, and high dose antibiotics and steroids were inadvertently injected into the anterior chamber and corneal stroma through a 19 g needle during the stromal hydration procedure for wound closure at the incision sites . The antibiotics and steroids given were gentamicin (20 mg/0.5 ml) and dexamethasone (2 mg/0.5 ml), originally prepared for subconjunctival injection . About 10 seconds after the injection, we recognized the mistake, and the anterior chamber was irrigated with 100ml of a balanced salt solution (cytosol ophthalmicstm) for one minute . On postoperative day one, the patient complained of blurred vision and foreign body sensations . Severe corneal edema and descemet's membrane folds invading the visual axis were seen extending from the 12 to 4 o'clock position (fig . Best - corrected visual acuity was 0.2, and intraocular pressure was 18 mmhg by noncontact tonometer . Postoperatively, cravit (levofloxacin 0.5%, santen, osaka, japan), 1% prednisolone, and 5% nacl were administered every two hours . Oral prednisolone (20 mg) and ofloxacin (300 mg) were used as well . A pressure patch with ofloxacin and dexamethasone ointment the corneal edema persisted until postoperative day three, and the best - corrected visual acuity decreased to 0.15 . The vision improved to 0.4 after two weeks, but the corneal edema persisted without improvement . The patient continued with the original and medications and dosages . On the third postoperative week, the best - corrected visual acuity stabilized at 0.4, and the corneal edema began to resolve . The visual acuity continued to improve to 0.7, and the corneal edema finally resolved four weeks postoperatively . However, the linear descemet's membrane folds persisted up to postoperative week 16 (fig . 2, 3). At the same site of the descemet's membrane fold, corresponding endothelial damage was also observed, manifesting as a definite dark acelluar area on specular microscopy (fig . 4). The preoperative endothelial cell density on specular microscopy was 2717 cells / mm and the mean cell size was 368 m . At postoperative month three, the mean cell size was 424 m and endothelial cell density had decreased to 2358 cells / mm . No other changes were observed after that, and the best - corrected visual acuity improved to 0.8 at postoperative month four . Almost every ocular drug can be potentially toxic to the corneal endothelium, and the risk of endothelial toxicity is increased when used in the anterior chamber because of its direct contact to the endothelium . Corneal endothelium is vulnerable even to osmotic change or ph change of the irrigating fluid, and is easily damaged by electrolyte imbalance . Moreover, permanent endothelial dysfunction, cystoid macular edema, pupil dilation, and glaucoma due to destruction of trabecular meshwork can occur and have been reported in severe cases.8 - 10 therefore, it is important to control the concentration of the solution when antibiotics are added to the irrigating solution in order to prevent postoperative endophthalmitis.1,2,4 however, the exact nontoxic concentrations of the antibiotics and steroids have not been fully investigated . Montan et al.11 studied doses and exposure durations of cefuroxime when injected in the anterior chamber, reporting that 1 mg/0.1 ml of cefuroxime is safe and maintains a high concentration for one hour postoperatively . Kang et al.12 studied gentamicin toxicity in different concentrations on three types of human corneal cells under culture and suggested that the safe local concentration of gentamicin for endothelial and stromal cell was 1 mg / ml in the human eye . With increasing concentration of gentamicin (from 2 mg / ml to 4 mg / ml), the toxicity of gentamicin to the corneal cells was in direct proportion to its concentration and duration of incubation . Petroutsos et al.13 reported that endothelial toxicity occurred when 1 mg / ml of gentamicin was injected into the anterior chamber in rabbit eyes . Medin14 used a weight recording system to demonstrate the possible toxic effect of gentamicin on the endothelium of rabbit corneas stored in organ culture . Lin et al.15 reported that 15% cell damage was found in tissue cultured bovine endothelial cells when exposed to 1.6 mg / ml of gentamicin . When the concentration of gentamicin was doubled, cell damage reached 40%, and the recovery of cellular morphology was much slower . Mester and stein16 studied the effect of gentamicin concentration on rabbit endothelial cells and reported that 0.50% gentamicin was toxic to endothelium, but 0.25% gentamicin was not significantly different as compared to the control . Besides irreversible damage to the corneal endothelium and corneal edema, high concentrations of gentamicin solutions can induce retinal hemorrhage, edema, and obliteration of the retinal vasculature when injected in the vitreous, resulting in optic atrophy, retinal pigment degeneration, or neovascular glaucoma.17,18 in our case, the toxicity of the gentamicin to the corneal endothelium was expected to be significant due to the fact that the concentration of the gentamicin injected in anterior chamber and corneal stroma was 40 times more than that of previously mentioned studies . Wang et al.19 tested the cytotoxicity of five antibiotics (amphotericin - b, colistin - m, sulbenicillin, amikacin, cephradine) and a steroid (betamethasone) to cultured porcine corneal endothelial cells and demonstrated that the steroid was less toxic to the endothelium than other antibiotics . In our case, the inadvertent injection of antibiotics and steroids resulted in severe corneal edema from 12 to 4 o'clock position involving the visual axis, along with extensive descemet's membrane folds . We used a hypertonic solution of about 1720 osm / l (5% nacl) topically to reduce the stromal edema . This concentration is known to be nontoxic,20,21 and the solution was effective in our case . On postoperative week 12, the descemet's membrane folds were observed by specular microscopy and persisted 16 weeks after the operation . In our case, corneal edema and the descemet's membrane fold were probably due to inadvertent corneal stromal injection with high dose gentamicin and dexamethasone rather than injection directly into the anterior chamber . The duration of exposure and the concentration of the drugs were negligible, due to prompt irrigation of the anterior chamber with balanced salt solution . We found the localized endothelial defect on specular microscopy manifesting as a definite dark acelluar area, which corresponded to the area of the descemet's membrane fold and site of inadvertent stromal injection . Also, irrigation with a balanced salt solution might play a role in causing corneal edema . On postoperative week four, the corneal edema resolved, and best corrected visual acuity improved to 0.7 . Our case showed relatively good visual prognosis despite the high concentration of the injected antibiotics and steroids . This favorable outcome might be due to the immediate irrigation of the anterior chamber with balanced salt solution . As such, long - term endothelial exposure to high dose antibiotics and steroid can be avoided . The immediate irrigation was possible because the balanced salt solution was prepared before the operation . Hansany and basu22 demonstrated that a 0.1 mg / ml concentration of gentamicin was not toxic to the corneal endothelium until seven days postop, but after seven days corneal endothelial toxicity was demonstrated for that concentration . This study suggested that duration of drug exposure (rather than drug concentration) is also an important factor for corneal endothelial damage . Therefore, considering our case and this study, we can conclude that immediate irrigation of the anterior chamber is important in preventing corneal endothelial damage when high dose antibiotics and steroids are injected in anterior chamber by mistake during cataract or other intraocular surgery . To dilute the intrastromal antibiotics and steroids, we considered stromal injection of a balanced salt solution, but this was not performed because separation of descemet's membrane from stroma can occur . Mcdonald et al.23 insisted that clear communication between the surgeon and nursing staff, in addition to precise labeling of every injectable solution, is necessary to prevent accidents, such as this . In addition, it is wise to draw up injectable antibiotics just before injection, and to use different gauge needles for the different solutions, which could easily be recognized by physician under the microscope . In our case, we reported a favorable visual outcome with immediate irrigation of the anterior chamber with a balanced salt solution after the inadvertent injection of high dose antibiotics and steroids . However, to prevent such an accident during surgery, strict precautions (mentioned previously) and clear communication between surgeons and nursing staff, including scrub nurses, are considered to be of great importance.

Autosomal - dominant polycystic kidney disease (adpkd) is characterized by cyst formation and occurs primarily in the kidneys . Due to the replacement of the normal renal parenchyma, adpkd results in end - stage renal failure in 45% of patients . Approximately 711% of patients receiving renal replacement therapy in the western world are doing so due to adpkd . The underlying cause of adpkd is a mutation in the polycystin-1 and -2 plasma proteins, located in the primary cilia . This mutation leads to abnormal function of renal tubular epithelia and inadequate calcium influx followed by cyst formation . Cardiovascular disease is the most frequent cause (36%) of mortality in patients with adpkd . Patients with adpkd often develop hypertension at an earlier age than the general population before any impairment of kidney function . It is hypothesized that the renin - aldosterone system and endothelial dysfunction caused by impaired nitric oxide release are the important factors in the development of hypertension in adpkd patients . We describe an uncommon case of a middle - aged man with a spontaneous coronary artery dissection (cad) and adpkd . A 41-year - old caucasian man presented at an emergency department with acute chest pain . The chest pain began 2 h before presentation . Medical history recorded a subdural haematoma, as a result of trauma . According to the family history, our patient was a non - smoker, with no history of hypertension, diabetes mellitus or hypercholesterolaemia . On physical examination, the blood pressure was 155/102 mmhg and the pulse 72 beats / minute; other vital parameters were normal . Electrocardiography showed a sinus rhythm of 65 beats / minute and st elevation in the precordial leads (figure 1). / l, creatine kinase mb was 17.4 u / l and troponin t was 0.21 ng / ml . The other laboratory test results were normal . Echocardiography revealed a hypokinetic septum and a slightly impaired left ventricular function with an ejection fraction of 4560% . Electrocardiography on presentation shows a sinus rhythm of 65 beats / minute and st elevation in v3, ii and iii . Based on these results, a presumptive diagnosis of acute septal myocardial infarction was made . Coronary artery angiography (cag) revealed a transient occlusion of the left anterior descending (lad) coronary artery, most probably as a result of myocardial bridging (figure 2a). The cag (a) shows the compression of the lad coronary artery during the systole resulting in narrowing and (b) performed after the second chest pain attack demonstrating a dissection in the distal left anterior descending coronary artery . However, the chest pain returned 3 days after the presentation . A second electrocardiography showed persistent inverted t waves in the precordial leads without st elevation . It disclosed an open lad with a dissection in the distal part and a double lumen, which was not observed during the first angiography (figure 2b). The definitive diagnosis of non - q - wave anterior infarct as a result of a spontaneous lad dissection was made . Based on the cysts in both kidneys combined with a family history of adpkd, the diagnosis of adpkd was made (ravine s criteria). Since cerebral aneurysms are one of the extrarenal manifestations of adpkd, a computerized tomography angiography of the brain was performed, with a negative result for vascular anomalies . An exercise stress test, performed 16 days after the onset of chest pain, was normal . The lad was patent without a significant infarction, but still with a double lumen appearance . At 2-year follow - up by a nephrologist, our patient, a relatively young man with a negative cardiovascular profile and a history of subdural haematoma, developed a myocardial infarction secondary to dissection of the lad . The cad was not recognized during the first cag because the relevant coronary artery can obturate the dissection in the acute phase . This condition is found when the segmental coronary artery has an intramyocardial course, being compressed during systole and restored during diastole . Therefore, one would require a difference in coronary artery contraction in the systolic and diastolic phase which was not observed on the first cag . Four cases were reported on the occurrence of a spontaneous cad in middle - aged adpkd patients . Predisposing factors for cad in adpkd patients are still undetermined, especially in the absence of traditional cardiovascular risk factors . Furthermore, it is not known if arterial dissection is an extrarenal manifestation of adpkd or if is just secondary to hypertension . The estimated prevalence of spontaneous cad is 0.7%, and it is in 2% of cases, the cause of acute coronary syndrome . The lad is affected in 80% of the patients with cad [8, 9]. The majority of patients with a cad often lack classical risk factors for cardiovascular disease and are female . De maio et al . Identified three groups of patients with cad: (i) patients with atherosclerotic cardiovascular disease, (ii) women in the postpartum period and (iii) an idiopathic group . Several underlying conditions in the idiopathic group are suggested, such as polyarteritis nodosa, lupus erythematosus, marfan s syndrome, ehlers danlos syndrome, intense physical exercise, the use of cocaine, cyclosporin and oral contraceptives . The tunica media, the middle layer of elastic arteries, include smooth muscles with interposing layers of elastic lamellae . The connection between the intracellular contractile filaments and extracellular elastic fibres is provided by dense plaque sites . Localization in the dense plaques assigns a significant function to polycystins in maintaining vascular integrity . Furthermore, qian et al . Suggested that abnormal intracellular calcium concentration in the vascular smooth muscle cells is linked to vascular phenotype in the case of inactivation of polycystin-2 protein . Literature also confirms the observation of intracranial aneurysms and myocardial infarction secondary to coronary aneurysms in certain families and the occurrence of vascular rupture and haemorrhage in homozygous polycystic kidney disease (pkd)-1 knockout mice [4, 11]. Considering these results, and the high plasma renin activity and impaired nitric oxide release in adpkd patients, we speculate that the vascular abnormalities are most likely a direct result of pkd mutations rather than a secondary cause of hypertension . In conclusion, polycystins seem to play a main role in the stability of the arterial vasculature . Therefore, a spontaneous cad should be considered as an extrarenal manifestation of adpkd . The clinician should be aware of cad if adpkd patients present with chest pain or discomfort . Cag can be inconclusive in respect to the mechanism of coronary occlusion in the acute phase and should be repeated, especially when the complaints persist.

Optimal treatment of cancers in children often requires combined - modality therapy, including: chemotherapy, surgery, and/or radiotherapy . Chemotherapy is not always sufficient to achieve the cure of solid tumors in children; either resection or radiation may be needed for local tumor control as well (1). Children with radiosensitive malignant tumors typically require radiation therapy for a number of sessions over a period of several weeks . Although the procedure is painless, young children need to be sedated or anesthetised in order to provide a motionless state during the procedure . A short period of sedation, analgesia or general anaesthesia the patient and anaesthesia equipment are observed continuously by closed - circuit television, and monitors are mirrored to the remote observation site outside the treatment room . Different anesthesia methods and anesthetics have been recommended to provide a safe and optimal situation of motionless with a short recovery period in children undergoing general anaesthesia or sedation for external beam irradiation (37). This report describes the accidental detection of a missed complication by the anesthetist at the time of radiotherapy, from the previous chemotherapy, which was preliminarily and unduly attributed to anesthesia . A 2.5 year - old, 13-kg boy, asa (american society of anesthesiologists) class ii, with acute lymphoblastic leukaemia (all), was scheduled for radiotherapy . Anesthesia was planned by the anesthetist, and the physical examination was normal before radiotherapy . Routine monitoring was established, and for peripheral pulse monitoring, the distal portion of the right upper limb of the child was rested out of the covers . After about 5 minutes, when the anesthetic effect of ketamine began, we noticed a lack of right radial pulse . Anesthesia - related hemodynamic instability was expected, however no other manifestation of hypoxia or hypo - perfusion was detected . Furthermore, carotid pulsation was normal, and examination of the left radial pulse and other peripheral pulses showed normal pulsation . The patient showed a scar and swelling on the right antecubital area . A more detailed history taken from the child s parents showed his history of chemotherapy during which extravasation of the chemotherapy drug had resulted in severe inflammation and edema at the site of injection . A colour doppler ultrasound of the antecubital area showed deep edema and chronic compression on the antecubital tissue and confirmed the diagnosis . Permission was obtained from the patient s parents to use the patient s reports, however the patient s information was to remain confidential . The incidence range of extravasations of cytostatic drugs in cancer patients has been reported from 0.2 to 1.4% in a five - year study (8). Extravasations in cytostatic treatment may cause a wide range of symptoms, from patients discomfort to severe complications such as necrosis and amputation . (9) eccrine squamous syringometaplasia, while rare, has also occurred in patients who have received chemotherapy treatment (10). Yeung et al described a case of metastatic ovarian carcinoma with repeated thrombosis of the femoral arteries following intravenous carboplatin - based combination chemotherapy . Persistent withdrawal occlusion (pwo) is frequently caused by fibrin sheath formation around venous access devices . Small doses of thrombolytic drugs (such as urokinase) could manage pwo, but could also serious complicate chemotherapy drug extravasation (12). Keratolytic ointment was applied for old lesions, whereas in new lesions, multiple subcutaneous injections of hydrocortisone solution were used before the application of betamethasone ointment . Application of conservative agents in radiotherapy - induced extravasation areas may avoid tissue necrosis and consequently, reconstructive surgery (13). Management of cytotoxic drug extravasation in humans is based on the experimental evidences and available case reports because of lack of randomized trials . For instance, topical dimethylsulfoxide (dmso) and cooling for extravasation of anthracyclines or mitomycin, local injection of hyaluronidase for extravasation of vinca alkaloids, and local injection of sodium thiosulfate (sodium hyposulfite) for extravasation of chlormethine (mechlorethamine; mustine) should be empirically recommended . In the case of failed conservative treatment, history and physical examination before injection of anesthetic agents could be useful in preventing patient mismanagement . A weak pulse in a child can be a significant problem for the anesthesiologist and in this case, it was shown to be a critically misleading factor . Increased emphasis is on clinical evaluation and pulse checking is necessary, especially in children with a history of chemotherapy . Providing thorough pulse evaluation when we work on children and avoiding immediate aggressive intervention before being certain about the cause of weak pulse are additional issues of importance shown by this case report.

Annually, more than 2.5 million muslims from about 140 countries go on the hajj pilgrimage, which is the largest mass gathering in the world . The presence of such a vast number of people in the holy places, especially in the cities of mecca and medina, and the massive pilgrim crowds in certain places at certain times, causes infectious diseases to spread more easily . Racial, cultural and health differences, as well as the level of access to health, medical and welfare services at the time of stay in these places are important factors that increase the risk of communicable diseases . Both, viral or bacterial respiratory tract infections are very common among pilgrims in all age and sex groups, and is regarded as the most common disease among pilgrims in some reports . Some studies have also reported the prevalence of different types of these infections up to 85.3% . To prevent these infections, some preventive measures have been recommended for the pilgrims, such as mandatory injection of tetravalent meningococcal meningitis vaccine, recommendation to inject seasonal flu vaccine, and using surgical masks . On the other hand, each country plans and implements some specific measures to prevent and control these diseases among pilgrims . More than 100,000 iranians in about 600 caravans undertook the hajj in 2010 . According to the current policies of the hajj medical center of iran, before traveling any caravan common diseases detected among pilgrims are treated in the caravan itself, or referred to the medical center, if needed . The reports show conflicting results about the effects of preventive measures for respiratory tract infections and in fact, a precise study on the effects of each one of these preventive measures has been rarely done . This research is trying to examine the effects of general preventive measures on respiratory tract infections through a nested case - control study, which is more precise design than ordinary case - control studies . We used nested case - control design, which is based on risk set sampling . The studied cohort consist two caravans (338 pilgrims), one of which was the first to enter mecca, and the other the first to enter medina . The outcome of this study was all types of respiratory tract infections other than the common cold including tonsillitis, pharyngitis, laryngitis, sinusitis, otitis media, bronchitis, pneumonia and influenza . As soon as any patient in the caravans was identified, a data collection form was completed for that individual . Considering that the level of presence in saudi arabia and the amount of contact with all types of viruses and bacteria are regarded as risk, on the day of identifying each patient, two pilgrims in the same caravan were randomly selected as control group . Each control pilgrim was not affected by the mentioned outcome at the time of investigation, although these pilgrims may be enrolled as a case in future days . Every pilgrim who become ill with studied disease and had consent to participate, was enrolled in the study . All pilgrims who did not get the studied disease at time of selection and had consent to participate, could be enrolled as controls . Body mass index (bmi) is calculated by dividing the weight in kilograms to height square in meter . If another patient with studied disease existed in the same room then the pilgrim considered as having room contacty . If the pilgrims used face masks in crowding, even occasionally were considered as using face mask . Pilgrims considered as vaccinated against influenza if they vaccinated by this vaccine since two years before traveling to saudi arabia . Pilgrims, who washed their mouth and throat with salt water at least once a day, were considered as having salt water gargling . Pilgrims with a past history of at least one systemic diseases including asthma, diabetes mellitus, hypertension, copd and cardiovascular diseases were considered as having systemic diseases . Present or past smoker with any type and amount of smoking were considered as smokers . If the pilgrims used personal prayer carpet in holy places were considered as using personal prayer carpet . The effect of each preventive measure on the occurrence of respiratory tract infections was studied through a univariable model . At the next stage, all variables which were significant in the first stage (p <0.2), were entered into the multiple models, and the adjusted odds ratio was obtained for each one of the variables . We used nested case - control design, which is based on risk set sampling . The studied cohort consist two caravans (338 pilgrims), one of which was the first to enter mecca, and the other the first to enter medina . The outcome of this study was all types of respiratory tract infections other than the common cold including tonsillitis, pharyngitis, laryngitis, sinusitis, otitis media, bronchitis, pneumonia and influenza . As soon as any patient in the caravans was identified, a data collection form was completed for that individual . Considering that the level of presence in saudi arabia and the amount of contact with all types of viruses and bacteria are regarded as risk, on the day of identifying each patient, two pilgrims in the same caravan were randomly selected as control group . Each control pilgrim was not affected by the mentioned outcome at the time of investigation, although these pilgrims may be enrolled as a case in future days . Every pilgrim who become ill with studied disease and had consent to participate, was enrolled in the study . All pilgrims who did not get the studied disease at time of selection and had consent to participate, could be enrolled as controls . Body mass index (bmi) is calculated by dividing the weight in kilograms to height square in meter . If another patient with studied disease existed in the same room then the pilgrim considered as having room contacty . If the pilgrims used face masks in crowding, even occasionally were considered as using face mask . Pilgrims considered as vaccinated against influenza if they vaccinated by this vaccine since two years before traveling to saudi arabia . Pilgrims, who washed their mouth and throat with salt water at least once a day, were considered as having salt water gargling . Pilgrims with a past history of at least one systemic diseases including asthma, diabetes mellitus, hypertension, copd and cardiovascular diseases were considered as having systemic diseases . Present or past smoker with any type and amount of smoking were considered as smokers . If the pilgrims used personal prayer carpet in holy places were considered as using personal prayer carpet . The effect of each preventive measure on the occurrence of respiratory tract infections was studied through a univariable model . At the next stage, all variables which were significant in the first stage (p <0.2), were entered into the multiple models, and the adjusted odds ratio was obtained for each one of the variables . During the hajj pilgrimage, 32 people (9.5%) of the cohort (338 pilgrims) were affected by respiratory tract infections . According to the explained method description of cases and controls, in terms of age, sex, education and other factors which influence the incidence of respiratory tract infections, are presented in table 1 . Distribution of factors affecting respiratory tract infections in hajj pilgrims, 2010 sixty nine percent of the patients were treated with symptomatic treatment and oral antibiotics, and 27.5% required treatment through intravenous antibiotics as well . In addition, 79.3% of the patients fully recovered through the treatment administered in the caravan, but 17.2% were referred to the hajj medical center, and finally 3.4% of the total patients were hospitalized in that center . Univariable logistic regression analysis showed that among the variables studied, only gargling with salt water, presence of other patients in the room, age and level of education, have p <0.2 and can be associated with respiratory tract infections . Among these variables, gargling with salt water with or = 2.4 and p = 0.08 had the strongest relationship [table 2]. The role of protective measures and other individual factors on respiratory tract infections among hajj pilgrims, 2010 the multivariable logistic regression model showed that none of the factors listed above were significantly associated with the outcome being studied . For example, presence of the other patient in the room increases the risk of outcome by 2.67 times, but this effect is not significant, according to the confidence intervals of or [table 2]. The results also showed that the current preventive measures including wearing face masks, seasonal flu vaccination, and use of personal prayer carpet were not effective enough in the prevention of these infections . However, the seasonal flu vaccination has been recommended, especially in high - risk individuals or those over 65 years of age . The influenza vaccine has been proved by other researchers to have no effect in the prevention of respiratory infections, and it has even been seen that the use of seasonal influenza vaccine prolongs the period of sore throat . However, methodological problems of the studies and the applied methods, including laboratory or demographic studies, should be considered when comparing these results . It should also be noted that recommendations to take a seasonal influenza vaccine is due to its preventive effect on the incidence of flu and reduction of the risk of pandemic swine flu . Other studies, like the results of this study, have shown that wearing face masks has no effect on respiratory infections . Although the cdc does not recommend wearing it, the saudi ministry of health recommends that pilgrims wear face masks . The recommendation to wear masks is probably because of its role in the prevention of tuberculosis, which has not been studied among the pilgrims . The effect of using a personal prayer carpet has not been studied in other reports . Probably, the close contact of the pilgrims with each other, and their presence in crowded places are the main causes for transmission of respiratory infections, so it hinders a possible protective role played by the personal prayer carpet . Although, the effect of this action on the prevention of respiratory infections was not significant in the multiple logistic model, it seems that the reason for it is the relatively small sample size in the study . In other words, through studies with a larger sample size, it may be concluded that lack of gargling with salt water increases the risk of respiratory infections by 2.3 times . Similarly, it can be said that staying with other patients in the same room increases the risk of respiratory infections by 2.67 times . As can be seen in table 2, factors such as age, sex, level of education, area of the room, smoking, systemic diseases, obesity, and the average daily hours of gathering together in the holy places have no effect on respiratory tract infections . In other words, any age and sex group with different individual conditions is likely to get exposed to viral and bacterial causes of these infections . As mentioned above, the relatively small sample size is one of the limitations of this study . An increase in the sample size would make it possible to study the role of factors associated with respiratory infections in any of the holy places (mecca, medina, mina, etc . ). In this study, the cases were of those who were diagnosed clinically by a caravan physician . If the rapid test diagnosis is applied along with the acceptable sensitivity and specificity to diagnose cases, the accuracy of the study will increase . Flu vaccinations wearing face masks, personal prayer carpet, smoking, obesity, etc ., do not affect the incidence of respiratory infections during the hajj, and if this study is to be undertaken with a larger sample size, according to the appropriate selected methodology, some evidence will be obtained showing the prophylactic effect of gargling with salt water in the prevention of these infections.

Care coordination is an important aspect of nursing care especially for elderly patients admitted to an acute care setting . In singapore care coordination and transitional care nursing is a new concept of care nevertheless important but unexplored . The objective of this paper is to explore the characteristics of elderly patients receiving care coordination, determine care gaps and intervention during home visit and telephonic review . A designed questionnaire was used to collect information on the patient s demography, social and clinical profile and determine post discharge activities using eric coleman s four pillars tool . The retrospective data from the patient s index admission from the last six months (nov 08april 09) was analyzed using spss version 16 . Majority, 69% were above 70 years old of which 57% female and 76% lives with their children . Clinical information demonstrates that 53% had 36 co - morbidities and 58% were taking more than five medications . The abbreviated mental test score were 6.2, 6% were depressed and delirium was present in 14% of patients . Only 65 patients (0.1%) had home visits and telephonic review done whilst 97% of the remaining had only telephonic review done . Those who had both telephonic and home visit review, medications advice and compliance were checked only in 0.8% (at one week) and 1.6% (at one month) whilst during home visit this was done in 12.2% of patients as medication discrepancy were apparent at home . As for appointment compliance and compilation were done in 0.8% at one week and 51% at four weeks of telephonic review compared to during home visit only 4.8% . Caregivers education was emphasized in 14% of patients at home visit, 2% at one week and 4% at one month of telephonic review . The result showed that home visit is effective in exploring medication compliance, advice and emphasizing caregiver education, managing appointments can be effectively done through telephone review . This study demonstrates the vital role of home visit for elderly patient to safely transit between hospitals to home.

The irrevocable aim of endodontics is a three - dimensional unblemished seal of the root canal system which is achieved by perfect designing of the canal diameter and canal form . The biomechanical preparation is one of the major steps for removal of bacteria and debris in the root canal so as to achieve a successful endodontic treatment . During root canal instrumentation there are complications such as perforations, ledge formation, transportation of canal, and formation of cracks in the root dentin . At times, in the zeal of biomechanical preparation of the canal we inevitably end up damaging the root dentin, which becomes a gateway to dentinal cracks and minute intricate fractures; thereby, causing failure of treatment . As a result of craze lines or microcracks, there might be occurrence of root fracture that propagates due to repeated application of stress by the occlusal forces . Shemesh et al ., observed more dentinal defects in teeth which were obturated with spreader than teeth obturated without spreader . In different degrees, dentinal damage can occur due to procedures like biomechanical preparation, obturation, and retreatment . Complexities in the preparation of root canal may be attributed to variation in the design of the cutting instrument, taper, or composition of the material from which it is made . Hand instrumentation the milestone of endodontic practice in the past though have lost popularity, still remain the integral part of canal preparation . Rotary instrument by its innate behavior in the canal may result in more friction, which may increase dentinal defects and microcracks formation in comparison to hand instruments . Possible relationship between the design of niti rotary instruments and the incidence of the vertical root fractures was found by kim et al ., and it was concluded that the design of the file affects strain concentration and the apical stress during instrumentation of root canal . Recently, the protaper next (ptn, dentsply, maillefer) files were introduced in the family of niti rotary instruments with a completely new design comprising of unique swaggering movement, greater flexibility, the m - wire technology, the 5 generation of continuous improvement, and its offset design . Whether it is rotary or hand files (hfs), they are assumed to cause limited frictional forces within the canal, hence creating dentinal defects . So there is need to study the behavior of different niti rotary instruments and the newly developed rotary system, ptn, on root dentin . Teeth with curved roots, calcified canals, extracanals, and teeth with developmental anomaly or resorption were excluded from the study . The teeth were decoronated at coronal portions by using a diamond disc, leaving roots approximately of 10 mm in length . All the roots were inspected with transmitted light for detecting any preexisting cracks or any craze - lines by using a stereomicroscope under 12, to exclude teeth with such findings from this study . Patency of the canal was established using a #10 k - file (mani, japan) in the canal . The specimens were then divided into four groups; each group containing 15 specimens each . Hfs upto file #40 were used for canal preparation . In the pt (dentsply, maillefer), hs (micro - mega, besancon, france), and ptn (dentsply, maillefer) groups; preparation of the canals was done using speed and torque controlled motor (x - smart; dentsply, maillefer). In the hand files group, step - back technique was used upto file #40 . In the pt group, the following sequence of pt rotary niti files were used for preparation of canals at 300 rpm: the shaping file x for coronal enlargement, and s1, s2, f1, f2, and f3 files, corresponding to apical size 30, used at the working length . In the hs group, the hs niti files were used upto file #30 at 300 rpm in crown - down sequence . In ptn group, the ptn rotary system files were used at 300 rpm in the following sequence: x1, x2 and x3, corresponding to apical size #30 . The ptn rotary files were used in a constant rotation at a speed of 300 rpm with light apical pressure (recommended torque is 2.0 ncm, adjustable up to 5.2 ncm according to practitioner experience). Flutes of the instruments were cleaned frequently to check any signs of distortion or wear . The ptn instruments are recommended to be used mechanically (manually in very severe curvatures) in a clockwise continuous motion with a brushing motion, away from external root concavities, to facilitate flute unloading and apical file progression . In all the experimental groups, sectioning of all the roots was done perpendicular to the long axis at 9, 6, and 3 mm using a diamond disc under water cooling . Digital images of each sectioned root was captured using a 40 stereomicroscope by using a digital camera (olympus, tokyo, japan). Roots were classified as no defect, fracture, and other defects as described in table 1 . Classification for identification of defects in the specimens the results were expressed as the number and percentage of defects in each group . Chi - square test was used for the statistical analysis of the groups . The level of significance was set at p = 0.05 using statistical package for social sciences (spss) 20.0 . Sectioning of all the roots was done perpendicular to the long axis at 9, 6, and 3 mm using a diamond disc under water cooling . Digital images of each sectioned root was captured using a 40 stereomicroscope by using a digital camera (olympus, tokyo, japan). Roots were classified as no defect, fracture, and other defects as described in table 1 . Classification for identification of defects in the specimens the level of significance was set at p = 0.05 using statistical package for social sciences (spss) 20.0 . Figure 1 is a bar chart representing the number of root defects in each group . Hfs group showed lowest defect (1/15) followed by pt (6/15), hs (10/15), and ptn (4/15). Statistical significant difference was seen between hfs and hs group and between hs and ptn groups (p <0.05). No significant difference was found between the pt and hs (p> 0.05). Bar chart representing number of root defects in each group the stereomicroscopic images of group i, ii, iii and iv are shown in figure 2 . Stereomicroscopic images showing dentinal defects seen in groups i, ii, iii, and iv, showing craze lines seen in group i. fracture and other defects in group ii craze lines and partial crack in group iii and craze lines in group iv in the present study; in hfs, pt, hs, and ptn, the number and incidence of defects observed in the root dentin was found to be 1/15 (6.67%), 6/15 (40%), 10/15 (66.67%), and 4/15 (26.7%), respectively . Group i (hfs) showed the lowest incidence of defects (6.67%); whereas, hs group showed the maximum incidence of defects (66.67%) as compared to other groups . The results of our study are in accordance with imam, who reported lowest number of defects (1/20) by hfs; and yold as et al ., observed highest incidence of defects (12/20) by hss rotary files ., in the present study the number and percentage of defects shown by the ptn rotary files were 4/15, that is, 26.7% . The results of the present study are not in accordance with the results by bier et al ., where the hfs showed no defect and pt rotary files showed highest incidence of dentinal damage (16%). Excess removal of root dentin during root canal preparation and obturation of the canal with spreader may create fracture in the teeth . The important goal in endodontics is resistance to tooth fracture because such fractures might cause decrease in the long - term survival rate . In the presents the number of rotations required for complete root canal preparation is more with niti rotary instruments than with the hfs . Kim et al ., stated that taper of the files is the responsible for increase of stress on the walls of the root canal; whereas, bier et al ., stated taper of the files as one of the contributing factor for crack formation in root dentin . Pt have more taper (0.07, 0.08, and 0.09, respectively) than the hss (0.04 and 0.06) and the ptn (x1, x2, and x3; 0.04, 0.06, and 0.07, respectively). This explains that there can be formation of cracks in the pt group, as reported earlier by bier et al ., liu et al ., barreto et al ., and liu et al . Furthermore, relatively low flexibility of the hs may have contributed to the maximum number of defects in hs group in the present study . Rotational force is applied to the canals of the root by niti rotary instruments, thus creating craze line or microcracks in root dentin . Formation of such defects may be associated with the design of tip, cross - sectional geometry, taper type (constant or progressive), flute form, and pitch (constant or variable). The pt files have a triangular cross - sectional geometry, hs having a triple helix cross - sectional geometry; whereas, the ptn is rectangular . Thus, it can be stated that design of the rotary files is not the only factor for defect formation in root dentin . Lam et al ., stated that forces shaping the root dentin can be affected by the file design . Risk of root fracture is increased due to the forces generated during the root canal preparation . Ptn files have m - wire technology with off - centered rectangular cross - section, giving the file a snake - like swaggering movement as it moves along the root canal, thus reducing the screw effect, the unwanted taper lock, and torque on any of the given file; thus decreasing the file - root dentin contact . M - wire alloy niti material with controlled memory niti wire are flexible than those made from conventional niti wire . Thus, such flexibility of ptn rotary files may have contributed in less number of dentinal defects formation as compared to pt and hs . Capar et al ., concluded that the swaggering motion and less taper of the ptn instruments could change the root canal volume to an extent as that of the higher tapered instruments . Use of different speed and torque settings for each rotary system could be the limitation of our study . Increase in the rotational speed is associated with increased cutting efficiency.simulation of periodontal ligament was not done in the present study . Capar i d et al . Stated that simulation of the periodontal ligament is necessary for investigating the influence of forces on formation of crack or fracture strength . It plays an important role in stress dissipation created by application of load to the teeth . Use of different speed and torque settings for each rotary system could be the limitation of our study . Capar i d et al . Stated that simulation of the periodontal ligament is necessary for investigating the influence of forces on formation of crack or fracture strength . It plays an important role in stress dissipation created by application of load to the teeth . Use of different speed and torque settings for each rotary system could be the limitation of our study . Increase in the rotational speed is associated with increased cutting efficiency.simulation of periodontal ligament was not done in the present study . Capar i d et al . Stated that simulation of the periodontal ligament is necessary for investigating the influence of forces on formation of crack or fracture strength . It plays an important role in stress dissipation created by application of load to the teeth . Use of different speed and torque settings for each rotary system could be the limitation of our study . Capar i d et al . Stated that simulation of the periodontal ligament is necessary for investigating the influence of forces on formation of crack or fracture strength . It plays an important role in stress dissipation created by application of load to the teeth . Within the limitations of this in vitro study, ptn rotary system can induce less dentinal defects than pt and hs.

Batch processes are used in production of many low - volume but high - value - added products (such as speciality chemicals, health care, food, agrochemicals, etc .) Because of operation flexibility in today's market - driven environment . In addition, if two or more products require similar processing steps, the same set of equipment is considered for at least economical reason . A batch plant producing multiple products is categorized as either a multiproduct plant or a multipurpose plant . In such a plant, all the products follow the same path through the process and only one product is manufactured at a time . Processing of other products is carried out using the same equipment in successive production runs or campaigns . In a multipurpose plant, each product follows one or more distinct processing paths; so more than one product may be produced simultaneously in such plants . The present work is directed toward the optimal design problems of multiproduct batch plants . In conventional optimal design of a multiproduct plant, production requirements of each product and a total production time for all products are available and specified . The number, the required volume, and size of parallel equipment units in each stage are then determined to minimize the investment . It should be emphasized that batch plantsdesign has been for long identified as a key problem in chemical engineering as reported in literature [29]. Formulation of batch plant design generally involves mathematical programming methods, such as linear programming (lp), nonlinear programming (nlp), mixed - integer linear programming (milp) or mixed - integer nonlinear programming (minlp). Mathematical programming or different optimization techniques, such as branch and bound, heuristics, genetic algorithm, simulated annealing, are thoroughly used to derive optimal solutions . However, in reality the multiproduct design problem can be formulated as a multiobjective design optimization problem in which one seeks to minimize investment, operation cost, and total production time, and, simultaneously, to maximize the revenue . Recall that not much work has been reported in the literature on the multiobjective optimal design of a multiproduct batch plant . Huang and wang introduced a fuzzy decision - making approach for multiobjective optimal design problem of a multiproduct batch plant . A monotonic increasing or decreasing membership function is used to define the degree of satisfaction for each objective function and the problem is then represented as an augmented minmax problem formulated as minlp models . To obtain a unique solution, presented the development of a two - stage methodology for multiobjective batch plant design and retrofit according to multiple criteria . The authors used a multiobjective genetic algorithm based on the combination of a single - objective genetic algorithm and a pareto sort procedure for proposing several plant structures and a discrete event simulator for evaluating the technical feasibility of the proposed configurations . In the case of multiple objectives, an optimum solution with respect to all objectives may not exist . In most cases, the objective functions are in conflict, because in order to decrease any of the objective functions, we need to increase other objective functions . Recently, solimanpur et al . Developed a sophisticated multiobjective integer programming model where the objectives considered were the maximization of total similarity between parts, the minimization of the total processing cost, the minimization of the total processing time and the minimization of the total investment needed for the acquisition of machines . The presence of multiple objectives in a problem usually gives rise to a family of nondominated solutions, largely known as pareto - optimal solutions, where each objective component of any solution along the pareto front can only be improved by degrading at least one of its other objective components . Since none of the solutions in the nondominated set is absolutely better than any other, any one of them is then an acceptable solution . As it is difficult to choose any particular solution for a multiobjective optimization problem without iterative interaction with the decision maker (dm) one general approach is to establish first the entire set of pareto - optimal solutions, where an external decision maker (dm) direct intervention gives interactive information in the multiobjective optimization loop . So, a satisfactory solution of the problem is found as soon as the knowledge is acquired . Promethee ii (preference ranking organisation method for enrichment evaluations2nd version) is a popular decision method that has been successfully applied in the selection of the final solution of multiobjective optimization problems . It generates a ranking of available points, according to the dm preferences, and the best ranked one is considered the favourite final solution . It is based on the concept of outranking relation, which is a binary relation defined between every pair (a, b) of alternatives, in such way that, if a is preferred to b (according to the dm interests), then it is said that a outranks b. when these relations are defined between all pairs of alternatives, they are exploited according to some rules in order to rank all solutions from the best to the worst . This is a nonpareto based approach based on the selection of several relevant groups of individuals, each group being associated to a given objective . It is reported that the method tends to crowd results at extremes of the solution space, often yielding to poor convergence of the pareto front . A more recent algorithm, based on scalarization with a weighted sum function, many successful evolutionary multiobjective optimization algorithms were developed based on the two ideas suggested by goldberg: pareto dominance and niching . Pareto dominance is used to exploit the search space in the direction of the pareto front and niching technique explores the search space along the front to keep diversity . The well - known algorithms in this category include multiobjective genetic algorithm: (moga), niched pareto genetic algorithm: (npga), strength pareto evolutionary algorithm: (spea), multiobjective evolutionary algorithm: (moea), the nondominated sorting genetic algorithm (nsga) proposed by srinivas and deb was one of the first evolutionary algorithm for solving multiobjective optimization problems . Although nsga has been successfully applied, the main criticisms of this approach has been its high computational complexity of nondominated sorting, lack of elitism, and need for specifying a tuneable parameter called sharing parameter . Recently, deb et al . Reported an improved version of nsga, which they called nsga - ii, to address all the above issues . The purpose of this study is to extend this methodology for solution of multiobjective optimal control problems under the framework of nsga - ii . The problem of multiproduct batch plant covered in this paper can be defined by assuming that the plant consists of a sequence of m batch processing stages that are used to manufacture n different products . At each stage j there are nj identical units in parallel operating out of phase, each with a size vj . Batches are transferred from one stage to the next without any delay, that is, we consider a zero - wait operating policy . In the conventional design of a multiproduct batch plant, one seeks to minimize the investment cost by determining the optimal number, required volume and size of parallel equipment units in each stage for a specified production requirement of each product and the total production time . However, in reality the designer considers not only minimizing the investment but also minimizing the operation cost and total production time while maximizing the revenue, simultaneously (1)maxnj, vj, bi, tli, qi, h revenue = f1=i=1ncpiqi, (2)minnj, vj, bi, tli, qi, hinvestment cost = f2=j=1mnjjvjbj, (3)minnj, vj, bi, tli, qi, h operation cost = f3=i=1n j=1mcejqjbi+coiqi, (4)minnj, vj, bi, tli, qi, h total production time = f4=h . So, the multiobjective problem consists of determining the following parameters: nj the number of parallel units in stage j, vj the required volume of a unit in stage j, bi size of the batch of product i at the end of the m stages, tli the cycle time for product i, qi the production requirement of product i and, h the total production time, while satisfying certain constraints such as volume, time, and so forth . Volume vj has to be able to process all the products i: (2) time constraint . The summation of available production time for all products is not more than the net total time for production (6)i=1nqibitlih . (3) the limiting cycle time for product i: (7)ijnjtli, i=1,,n; j=1,,m . Every unit has restricted allowable range (8)vjlvjvju, j=1,,m, bjlbjbju, j=1,,n . In contrast to the simple genetic algorithms that look for the unique solution, the multiobjective genetic algorithm tries to find as many elements of the pareto set as possible . For the case of the nsga - ii, this one is provided with operators who allow it to know the level of nondominance of every solution as well as the grade of closeness with other solutions; which allows it to explore widely inside the feasible region . In a brief form, the functioning of the multiobjective genetic algorithm nsga - ii can be described through the following steps . Fast nondominated sorta very efficient procedure, is used to arrange the solutions in fronts (nondominated arranging), in accordance with their aptitude values . A domination count np, the number of solutions which dominates the solution p, and a set (sp), that contains the solutions that are dominated for p. the solutions of the first front have the higher status of nondominance in the pareto sense . A very efficient procedure, is used to arrange the solutions in fronts (nondominated arranging), in accordance with their aptitude values . A domination count np, the number of solutions which dominates the solution p, and a set (sp), that contains the solutions that are dominated for p. the solutions of the first front have the higher status of nondominance in the pareto sense . Diversity preservationthis is achieved, by means of the calculation of the crowding degree or closeness for each of the solutions inside the population . This quantity is obtained, by calculating the average distance of two points on either side of a particular solution along each of the objectives . This quantity serves as an estimate of the cuboid perimeter, formed by using the nearest neighbours as the vertices . There is also, an operator called crowded - comparison (n), which guides to the genetic algorithm, towards the pareto optimal front, in accordance with the following criterion: (9)in j if (irank <jrank),or (irank = jrank) and (ididtance> jdistance). In accordance with the previous criterion, between two nondominated solutions, we prefer the solution with the better rank . Otherwise, if both solutions belong to the same front, then, we prefer the solution that is located in a lesser crowded region . This is achieved, by means of the calculation of the crowding degree or closeness for each of the solutions inside the population . This quantity is obtained, by calculating the average distance of two points on either side of a particular solution along each of the objectives . This quantity serves as an estimate of the cuboid perimeter, formed by using the nearest neighbours as the vertices . There is also, an operator called crowded - comparison (n), which guides to the genetic algorithm, towards the pareto optimal front, in accordance with the following criterion: (9)in j if (irank <jrank),or (irank = jrank) and (ididtance> jdistance). In accordance with the previous criterion, between two nondominated solutions, we prefer the solution with the better rank . Otherwise, if both solutions belong to the same front, then, we prefer the solution that is located in a lesser crowded region . Initial loopinitially, a random parent population (po) of size n is created . Then the usual binary tournament selection, recombination and mutation operators are used to create a new population (q0), of size n. initially, a random parent population (po) of size n is created . Then the usual binary tournament selection, recombination and mutation operators are used to create a new population (q0), of size n. main loopthe nsga - ii procedure can be explained, by describing the th generation just as it is showed in figure 1 . The procedure begins with the combination of pt and qt forming a new population called rt, then the population rt is sorted using the nondomination criterion . Since all previous and current population members are included in rt, the population rt has a size of 2n, later, the different fronts of nondominated solutions are created, being f1 the front that contains the better rank solutions . Figure 4 shows that, during the process of forming the new population pt+1, the algorithm takes all members of the fronts f1 and f2, and some elements of the front f3; this is, because n solutions are needed exactly for the new population pt+1 to find them exactly n solutions, the last front is ordained, which for this description is the number 3, arranging the solutions in descending order by means of the crowded comparison (n), and selecting the best solutions needed to fill all population slots . After having the population pt+1, the genetic operators of selection, crossing and mutation, are used to create the new population qt+1 of size n. finally it is mentioned that the selection process, the crowded comparison operator is used . The nsga - ii procedure can be explained, by describing the th generation just as it is showed in figure 1 . The procedure begins with the combination of pt and qt forming a new population called rt, then the population rt is sorted using the nondomination criterion . Since all previous and current population members are included in rt, the population rt has a size of 2n, later, the different fronts of nondominated solutions are created, being f1 the front that contains the better rank solutions . Figure 4 shows that, during the process of forming the new population pt+1, the algorithm takes all members of the fronts f1 and f2, and some elements of the front f3; this is, because n solutions are needed exactly for the new population pt+1 to find them exactly n solutions, the last front is ordained, which for this description is the number 3, arranging the solutions in descending order by means of the crowded comparison (n), and selecting the best solutions needed to fill all population slots . After having the population pt+1, the genetic operators of selection, crossing and mutation, are used to create the new population qt+1 of size n. finally it is mentioned that the selection process, the crowded comparison operator is used . To demonstrate the effectiveness of nsga - ii on batch plant processes two examples are given here . The first example is about a batch plant consisting of three processing stages (mixer, reactor, and centrifuge) to manufacture two products, a and b. the second example treats four processing stages (mixer, reactor, extraction and centrifuge) to manufacture three products a, b and c. the data for examples 1 and 2 are illustrated, respectively, in tables 1 and 2 (the processing times, size factor for the units and cost for each product). The set of decision variables consists of the batch size, the total production time, the number of parallel units at each stage, the cycle time for each product, and the required volume of a unit in each stage . Since the number of parallel units at each stage is an integer decision variable, we code this variable as a binary variable . In addition to the constraints expressed by (5)(8), we consider bounds on objective functions as additional constraints to generate feasible nondominated solutions in the range desired by the decision - maker, to have 19 constraints in all (10)filfifiu, i = 1,4 . Then nsga - ii is employed to solve the optimization problem with the following parameters: maximum number of generation up to 200, population size 500, probability of crossover 0.85, probability of mutation 0.05, distribution index for the simulated crossover operation 10 and distribution index for the simulated mutation operation 20 . The revenue (f1) increases with the increase in operation cost (f3), while the investment cost (f2) decreases . When all the four objective functions are considered simultaneously, solutions obtained in the present study show improvement as by huang and wang results for the same problem . For example, let us consider the solution presented by huang and wang with unit reference membership level for all objectives: f1 = 121 350, f2 = 171 624, f3 = 77 299, f4 = 5667 . The solution (1) presented in table 3 of the present study improves the above solution f1, f3, f4 while f2 is comparable . The large set of multiple optimal solutions provides the decision maker with immediate information about the relationship among the several objective criteria and a set of feasible solutions . Thus, it helps the decision - maker to select a highly confident choice of solution . The fixed optimal plant structure as 221 corresponds to a two mixers, two reactors, and one centrifuge design . The batch plant in this case consists of four processing stages to manufacture three products a, b, and c, with four - objective optimization problem as expressed in (1)(4). But we deal with 4 integer variables, 14 real variables, and 31 constraints which includes bounds on objective functions . The same model equations of example 1 are used here except the processing time, ij in (7). The time required to process one batch of product i in stage j is expressed as: (11)ij=ij+cijbij, i, j, where ij 0, cij 0 and j are constants and bi is the batch size for product i. thus, the processing time is not a constant, but depends on the decision parameters of the batch size . The constrained multiobjective minlp problem is solved by nsga - ii with the same set of nsga - ii parameters as used in example 1 . As in example 1, the revenue (f1) increases as operation cost (f3) increases, while the investment cost (f2) decreases following operation cost (f3). Let mention that when all four objective functions are considered simultaneously, the solutions obtained in the present study improve significantly the results presented by huang and wang for the same problem . For example, the solution presented by huang and wang with unit reference membership level for all objectives (f1 = 274 312, f2 = 375 688, f3 = 175 688, f4 = 5639) the solution (1) presented in table 4 of the present study improves the above solution f1, f3, f4 while f2 is comparable . In this example, the plant structure evolved as optimal is: two mixers, two reactors, two extractors, and one centrifuge as presented in figure 7 . Implementation of a trade - off analysis is dependent upon the availability of the decision - maker's preferences . A multiobjective decision in a batch plant process design is considered and a non dominating sorting genetic algorithm (nsga - ii) is developed to get an optimal zone containing solutions under the concept of pareto set . Nsga - ii capability has been proved in evolving the entire set of nondominating solutions along the pareto front in a single run of the algorithm . Thus, the decision maker (dm) is provided with the best trade - off operating zone . Furthermore, a better confident choice of design among several compromises of the decision maker can be achieved if the decision variables effects on the objective functions are known . Finally, the large set of solutions presents a useful base for further alternative approaches to fulfil the dm targets . The inherent dynamic nature of batch processes allows for their ability to handle variations in feedstock and product specifications and provides the flexibility required for multiproduct or multipurpose facilities . They are thus best suited for the manufacture of low - volume, high - value products, such as specialty chemicals, pharmaceuticals, agricultural, food, and consumer products, and most recently the constantly growing spectrum of biotechnology - enabled products . Reduced time to market, lower production costs, and improved flexibility are all critical success factors for batch processes.

Since the introduction of extra - oral implants in reconstruction of craniofacial defects, achieving proper prosthesis retention has become more promising . These problems include ulceration of hard and/or soft tissues used for retention, lack of retention due to prosthesis movement, and tissue irritation caused by adhesives . The ideal position and number of implants for restoring orbital defects would be three non - linear implants in lateral, supraorbital, and infra - orbital rims . However, such implant arrangement is not always conceivable considering the extension of the defect, and bone quality and quantity of defect s walls . Two of the most common retention systems used in reconstruction of orbital defects include freestanding abutments with magnetic retention and bar - clip retention . Magnetic abutments are more common because they resolve the potential problems associated with bar - clip attachment including difficulty in insertion and removal of prosthesis by the patient, difficulty in regular hygiene measurements, and rigidity of the attachment resulting in implant overloading . However, magnetic attachment might not provide sufficient retention if implants have been placed adjacently . The presence of implant in the defective area might complicate the usual impression - taking procedures used in fabrication of conventional craniofacial prostheses . Accuracy of the impression is affected by defect shape, retention system, number, and divergence of the implants . Moreover, anatomical undercuts in the defect, and proximity or remoteness of the implants could complicate the impression - taking procedure . Use of multiple trays, elastomeric impression materials, and dual impression technique have been suggested to overcome such problems [2,1214]. The purpose of this article was to present a case treated with an implant - supported prosthesis to reconstruct a relatively large orbital defect using three adjacent implants in the lateral orbital rim . A 60-year - old woman with a left orbital defect due to removal of periocular basal cell tumor was referred to the implant department of tehran university of medical sciences, school of dentistry, for prosthetic reconstruction of the eye . Three implants (superline, dentium, seoul, south korea), 8 mm in length and 3.6 mm in diameter were placed in the lateral rim of the orbit . Although the most suitable sites for orbital implants are the superior and lateral rims, in the present case the implants have been placed adjacently, due to insufficient bone thickness in superior and inferior orbital rims . The defect was relatively deep with undercuts in the medial wall which could complicate impression making . The preferred prosthesis design was an implant - supported prosthesis with a custom bar containing properly distributed magnetic components . The healing abutments were unscrewed and three hexed direct - casting abutments (implantium, dentium, seoul, south korea) with 4.5 mm diameter were directly secured to the implants . The medial undercuts were blocked out, using a gauze pack to avoid the penetration of acrylic resin . An auto - polymerizing acrylic resin (pattern resin, gc, tokyo, japan) pattern was formed directly on the abutments in a manner that cobalt samarium (co5sm) magnets (implantium, dentium, seoul, south korea), with 5.5 mm diameter and retention force of 700 gram could be placed at proper distances in the superior, inferior and lateral segments of the acrylic bar (fig . The acrylic resin bar was casted using base metal alloy (aalba dent inc . ; cordelia, c.a, usa) and the magnet keepers were cemented in corresponding sites with panavia f 2.0 resin cement (kurary medical inc, japan). Acrylic resin pattern of bar containing indentations for magnets (a), try - in of metal bar on the implants with magnet keepers in place (b). The space beneath the superstructure and also the undercuts in defect walls were blocked out with gauze packs . The final impression was made in order to pick up the magnets and simultaneously record the rest of the orbital defect . Light viscosity addition silicone (panasil, kettenbach, germany) was used as the first layer to cover the entire defect as well as the intact side of the midface . Afterwards, regular viscosity addition silicone (panasil, kettenbach, germany) was used over the light viscosity material to create mechanical retention projections for the gypsum layer (herostone vigodent inc . The wax pattern of the orbit was formed containing an ocular prosthesis which simulated the properties of a healthy eye . The pattern was tried on the patient and some modifications were made to improve its esthetic and adaptation . The prosthesis was made of a combination of heat - cured acrylic resin for holding the magnets, and high - temperature vulcanizing silicone with internal / external staining and other characterizations of the skin, such as wrinkles, eye brow and eye lashes . The final prosthesis was delivered to the patient and necessary home care instructions were provided such as removing the prosthesis during night, cleaning the eye defect with damp gauze, and the need for regular biannual follow - ups [69]. Tissue side of the prosthesis with three magnets (a), delivery of the prosthesis (b) the patient presented here has been treated with an implant - supported orbital prosthesis with bar - magnetic attachment . This retention mechanism might minimize the risk of mechanical overload on the implants compared to a conventional bar - clip attachment with cantilever arms . Despite the proximity of implants, the mentioned distribution of magnetic attachments has increased the retention through creating a tripod . Furthermore, since the acrylic resin pattern of the bar was made directly in the defective area, no implant or abutment analogues were used in final impression procedure . Prolonged chair - side time is a disadvantage of the stated method which could be justified considering the mentioned advantages.

Hepatocellular carcinoma (hcc) is one of the most common cancers worldwide especially in china [13]. The high hcc rates in china largely reflect the elevated prevalence of chronic hepatitis b virus (hbv) infection . In developed countries such as united states, nonalcoholic fatty liver disease (nafld), associated with obesity, less study focused on the effect of obesity on hcc especially in the relationship between bmi and hcc . No study has focused on the effect of waistline on the surgical outcome of hcc patients . So, this study is about the influence of body mass index (bmi) and waistline on complications, postoperative death and long - time survival in patients undergoing surgery for hcc . Between january 2007 and december 2012, we retrospectively reviewed the database of 136 patients underwent hepatic resection for hcc at the department of surgery of the fourth affiliated hospital of zhejiang university school of medicine . This retrospective study had been performed with the approval of the ethics committee of the fourth affiliated hospital of zhejiang university school of medicine and was in compliance with the helsinki declaration . Written informed consent was given by participants for their clinical records to be used in this study . We enrolled 136 patients and divided them into 4 groups: group a, bmi <25; group b, bmi 25; group c, waistline <90 cm in males or waistline <80 cm in females; group d, waistline 90 cm in males or waistline 80 cm in females . There were 61 patients (44.9%) in group a, 75 patients (55.1%) in group b, 58 patients (42.6%) in group c, and 78 patients (57.4%) in group d. all patients followed the same preoperative evaluation protocol, including blood biochemistry, ultrasonography, and computed tomography of the liver . The criteria for resection included good general condition, absence of essential organ dysfunction and distant metastasis, and liver function (child a or b, icg r15 <15%). Follow - up data were obtained by direct communication with patients after they underwent hepatic resection . Survival analysis, including overall survival and disease - free survival, was estimated by the kaplan - meier survival method and compared using the log - rank test . All statistical analyses were performed using statistical software (spss 13.0 for windows, spss, chicago, il) at a significant level of p<0.05 . There were 110 (80.9%) men and 26 (19.1%) women who underwent hepatectomy . The median age was 43 years; 118 (86.8%) patients were ages <60 years and 18 (13.2%) patients were older than 60 years . There were 61 (44.9) patients with normal bmi (<25) and 75 (55.1%) patients were overweight or obese (bmi 25). Fifty - eight patients (42.6%) belong to group c and 78 patients (57.4%) belong to group d. ninety - seven (71.3%) patients had positive hepatitis b and 39 (28.7%) patients without hbv . Histopathology of the hcc confirmed vascular invasion in 41 patients (30.1%) and liver cirrhosis in 65 (47.8%) patients . Surgical results confirmed capsular formation in 54 (39.7%) patients and 51 (37.5%) patients with solitary hcc (table 1). The intraoperative and postoperative data including major complications of 136 patients with hcc are shown in table 2 and supplementary table 1 . The median hospital stay was 11 days in group a and 12 days in group b, with no significant difference between the 2 groups (p=0.373). A, 23 (37.7%) patients had blood loss 1000 ml compared to 29 (38.7%) patients in group b. there was no hospital death in group a and 1 (1.3%) hospital death in group b. however, no significant difference was found between the 2 groups . There were no significant differences in overall postoperative complication rate between group a and group b, although pulmonary infection showed a significant difference between these 2 groups (p=0.017). Seven (11.5%) patients had complications in group a compared to 9 (12%) patients in group b. the most common complications in group a were ascites and wound infection, while the most common complications in group b were pulmonary infection and wound infection . Variables that might affect overall survival of patients with hcc after hepatic resection were analyzed in this study . On univariate analysis of the prognostic factors, we found that patients with bmi 25, waistline 90 cm (or 80 cm in women) and vascular invasion had poor overall survival than that without these variables (p=0.032, p=0.017, p=0.009). The other variables such as sex, afp level, and blood loss were not the significant prognostic factors of overall survival . On multivariate analysis, however, only vascular invasion and waistline were significantly associated with overall survival (p=0.031, p=0.039). The other variables, including bmi, were not independent prognostic factors of overall survival on multivariate analysis (table 3). The overall survival curves of hcc patients according to the bmi are shown in figure 1 . The 1-, 3-, and 5-year overall survival rates of group a were 95%, 81%, and 16%, respectively . The 1-, 3-, and 5-year disease - free survival rates of these patients were 78%, 38% and 4%, respectively (figure 1). However, there was no significant difference between the 2 groups in disease - free survival (p=0.235). The overall and disease - free survival curves of hcc patients affected by waistline were shown in figure 2 . The 1-, 3-, and 5-year overall survival rates of group c were 97%, 88%, respectively, and 15% compared to 96%, 68%, 15%, respectively, in group d. the 1-, 3-, and 5-year disease - free survival rates of group c were 84%, 40% and 6%, respectively . Group c had significant better overall survival and disease - free survival than group d (p=0.028, p=0.048). There were 110 (80.9%) men and 26 (19.1%) women who underwent hepatectomy . The median age was 43 years; 118 (86.8%) patients were ages <60 years and 18 (13.2%) patients were older than 60 years . There were 61 (44.9) patients with normal bmi (<25) and 75 (55.1%) patients were overweight or obese (bmi 25). Fifty - eight patients (42.6%) belong to group c and 78 patients (57.4%) belong to group d. ninety - seven (71.3%) patients had positive hepatitis b and 39 (28.7%) patients without hbv . Histopathology of the hcc confirmed vascular invasion in 41 patients (30.1%) and liver cirrhosis in 65 (47.8%) patients . Surgical results confirmed capsular formation in 54 (39.7%) patients and 51 (37.5%) patients with solitary hcc (table 1). The intraoperative and postoperative data including major complications of 136 patients with hcc are shown in table 2 and supplementary table 1 . The median hospital stay was 11 days in group a and 12 days in group b, with no significant difference between the 2 groups (p=0.373). A, 23 (37.7%) patients had blood loss 1000 ml compared to 29 (38.7%) patients in group b. there was no hospital death in group a and 1 (1.3%) hospital death in group b. however, no significant difference was found between the 2 groups . There were no significant differences in overall postoperative complication rate between group a and group b, although pulmonary infection showed a significant difference between these 2 groups (p=0.017). Seven (11.5%) patients had complications in group a compared to 9 (12%) patients in group b. the most common complications in group a were ascites and wound infection, while the most common complications in group b were pulmonary infection and wound infection . Variables that might affect overall survival of patients with hcc after hepatic resection were analyzed in this study . On univariate analysis of the prognostic factors, we found that patients with bmi 25, waistline 90 cm (or 80 cm in women) and vascular invasion had poor overall survival than that without these variables (p=0.032, p=0.017, p=0.009). The other variables such as sex, afp level, and blood loss were not the significant prognostic factors of overall survival . On multivariate analysis, however, only vascular invasion and waistline were significantly associated with overall survival (p=0.031, p=0.039). The other variables, including bmi, were not independent prognostic factors of overall survival on multivariate analysis (table 3). The overall survival curves of hcc patients according to the bmi are shown in figure 1 . The 1-, 3-, and 5-year overall survival rates of group a were 95%, 81%, and 16%, respectively . The 1-, 3-, and 5-year disease - free survival rates of these patients were 78%, 38% and 4%, respectively (figure 1). However, there was no significant difference between the 2 groups in disease - free survival (p=0.235). The overall and disease - free survival curves of hcc patients affected by waistline were shown in figure 2 . The 1-, 3-, and 5-year overall survival rates of group c were 97%, 88%, respectively, and 15% compared to 96%, 68%, 15%, respectively, in group d. the 1-, 3-, and 5-year disease - free survival rates of group c were 84%, 40% and 6%, respectively . Group c had significant better overall survival and disease - free survival than group d (p=0.028, p=0.048). There are many reports about the relationship between bmi, the size of waistline, and cancer risk [714]. Research conducted by the cancer council of victoria, australia shows that a waist measurement of over 100 cm for men and over 85 cm for women can significantly increase the risk of cancer including breast, bowel, and aggressive prostate cancer . However, there were few reports about the relationship between bmi, waistline, and hcc . The high hcc rates in china are largely due to the prevalence of chronic hepatitis b virus infection . However, with the increase in prevalence of obesity, nafld, which can progress to cirrhosis and subsequently to the development of hcc, is now recognized as one of the most common liver diseases in china . Although many reports had affirmed that patient with higher bmi tend to show a higher incidence of liver cirrhosis, there are few reports about the relationship between the bmi and the surgical outcome of hepatectomy for hcc [1517]. Regarding the relationship between bmi, waistline, and obesity, we hypothesized that waistline might be related to surgical outcome of hepatectomy for hcc, as well . In this study, there were no significant differences in postoperative complication rate and postoperative death between group a and group b. however, pulmonary infection (13.3%) showed a significant difference between the 2 groups (p=0.017). The observation that the obese patients had more pulmonary infection after hepatectomy for hcc can be explained by the fact that obesity can reduce pulmonary function through diminishing exercise capacity and lung volume, and increasing the resistance of breathing . Vascular invasion, waistline, and bmi are the independent prognostic factors for long - term survival . Group c had better overall and disease - free survival than group d, and group a had the better overall survival than group b. this result showed that bmi and waistline are both independent prognostic factors for long - term survival . Waistline may be more important than bmi in predicting the prognosis of hcc after hepatectomy . Obesity, which can lead to nafld and finally progress to cirrhosis, is thought to account for the majority of hcc [1821]. With the increase in prevalence of obesity, we recommend that individuals whose bmi or waistline exceeds normal limits should cultivate a healthy lifestyle, such as eating foods that help to lose weight, regular physical exercise, and getting enough sleep . In conclusion, overweight and obesity have been shown to be significant predictors of an adverse long - time survival after hepatectomy for hcc . Waistline is more important than bmi in predicting the disease - free survival of hcc after hepatectomy.

Professional mononuclear phagocytes, such as polymorphonuclear neutrophils (pmn), monocytes, and macrophages, are considered as the first line of defence of the early host innate immune response [1, 2]. Their main function has been classically understood to kill invasive pathogens by a variety of potent intracellular microbicidal effector mechanisms [37]. After the first contact with pathogens, mononuclear phagocytes engulf and internalize them into their phagosomes . By the fusion with intracellular granules and the formation of phagolysosomes the pathogens may be killed intracellularly by a combination of non - oxidative and oxidative mechanisms [1, 8]. Actions of potent antimicrobial peptides, such as defensins, cathelicidins, cathepsins, pentraxin, and lactoferrin, are parts of non - oxidative killing mechanisms, while oxidative killing relies exclusively on the production of antimicrobial reactive oxygen species (ros) via the nadph oxidase (nox) complex . Within blood circulating phagocytes, pmn are by far the most abundant cell population representing 5080% of the total white blood cells in different vertebrates . Moreover, after being released from the bone marrow into the blood circulation, pmn are highly mobile and short - lived phagocytes, being densely packed with secretory granules [4, 8]. Pmn granules are categorized into three different types based on their contents: primary (azurophilic), secondary (specific), and tertiary (gelatinase) granules . The types of granules to be found in circulating pmn depend on their maturation stage . Thus, pmn maturation starts with the formation of primary granules, followed by secondary and tertiary granules [4, 9, 10]. The content of primary granules includes myeloperoxidase (mpo), neutrophil elastase (ne), cathepsin g, proteinase 3, defensins, and lysozyme; secondary granules contain collagenase, gelatinase, cystatin, lysozyme, and lactoferrin; and tertiary granules comprise gelatinase, lysozyme, and arginase amongst others . Following granule maturation, pmn will possess all three types of granules displaying full killing capacity not only in the blood but also in tissues / organs and gut lumen . In addition, pmn act against pathogens by actively participating in complex inflammatory networks such as the release of a broad panel of proinflammatory chemokines, cytokines, and survival- and growth - factors which trigger both downstream proinflammatory effects and the transition into adaptive immune reactions . As such, several proinflammatory cytokines / chemokines were found enhanced in activated pmn in response to apicomplexan parasites, such as tnf-, il-1, cc, and cxc chemokines (e.g., il-8, ip-10, gro-, rantes, and mip-1) [1115]. Several of pmn - derived immunomodulatory molecules can augment the production of various chemokines and cytokines to further regulate phagocyte functions [16, 17]. More importantly, by this means activated pmn recruit not only other innate immune cells but also t cells to the site of infection [1820] or even induce sterile inflammation [21, 22]. Beginning with the landmark study of brinkmann et al ., the paradigm of how pmn fight and kill pathogenic bacteria has profoundly been changed . The discovery of dna - based antimicrobial neutrophil extracellular traps (nets) changed the current knowledge on innate immune reactions not only on the level of the pathogen killing but also on the pathophysiology of metabolic, autoimmune, reproductive, and inflammatory diseases, as well as cancer progression [3237]. Nets are released by activated pmn by a novel cell death process, called netosis, which can be stimulated by a variety of molecules and invasive pathogens . Microorganisms such as bacteria [31, 3941], fungi [4244], viruses [4549], and parasites [5055] were identified as net inducers . Also different molecules or cellular structures such as gm - csf / complement factor 5a [56, 57], activated platelets [40, 58], pma and zymosan [24, 26, 31, 59], singlet oxygen, lps [31, 61], and fc receptor trigger netosis . In addition, il-8 as well - known chemoattractant for pmn was demonstrated as net inducer [31, 62]. Efficient netosis requires mature pmn and in most cases nox, mpo, ne, and peptidylarginine deiminase type iv (pad4) activities [14, 24, 59, 6365]. Furthermore, the process of netosis obviously requires intracellularly signalling pathways of which raf - mek - erk kinases as well as p38 mapk are being the most frequently reported to be involved in this process [14, 23, 33, 6669]. In addition, calcium release is needed for optimal net formation in different vertebrate species [14, 23, 7072]. Upon stimulation of pmn, the nuclear envelope disintegrates permitting the mixture of chromatin with granular proteins / peptides . Ne and mpo degrade histones (h1, h2a / h2b, h3, and h4) and promote chromatin decondensation, mediated by pad4 via hypercitrullinating of specific histones to allow electrostatic coiling of the chromatin [64, 73, 74]. The total of the dna complexes being decorated with granular proteins / peptides and specific histones (h1, h2a / h2b, h3, and h4) are finally extruded as nets to the extracellular environment by dying pmn . Net formation is primarily a nox - dependent mechanism [14, 24, 59, 75, 76]. However, nox - independent netosis was also reported [29, 60, 67, 68, 77]. This mode of netosis is accompanied by a substantially lower level of erk activation and rather moderate level of akt activation, whereas activation of p38 is similar in both kinds of net formation [67, 68]. As an example irrespectively of nox - dependency, pathogens may either be immobilised within sticky dna fibres [55, 78, 79] or be killed via the local high concentration of effector molecules [31, 42, 51, 53]. Meanwhile, other types of leukocytes of the innate immune system, such as macrophages [8083], monocytes [26, 28], mast cells [84, 85], eosinophils [55, 86, 87], and also basophils, have been reported to release net - like structures which are now collectively entitled as extracellular traps (ets). Described already many years ago that enucleated pmn may remain vital and are even capable of killing invasive microbes . More recent studies corroborated these findings proving that leukocytes do not necessarily die after et extrusion [56, 68, 86]. In this context, yousefi et al . [56, 86] demonstrated that eosinophils and certain pmn subpopulations release ets of mitochondrial origin without dying . Furthermore, yipp et al . Verified that pmn which had released nets were still viable and retained their capability to engulf bacteria via phagocytosis . However, it appears to be nonlethal for pmn and faster than nox - dependent net formation and to rely on a vesicular - based pathway releasing nuclear dna [33, 68]. Additionally, different molecular pathways will lead in a stimulus - dependent manner to the extrusion of different types of ets in vitro and in vivo . Different morphological forms of ets were for the first time described in the human gout disease in vivo proving that monosodium urate crystals (msu) induced aggregated (aggets), spread (sprets), and diffused (diffets) et formation . As such, the parasitic nematode haemonchus contortus larvae triggered in ruminant pmn and eosinophils aggets, spreets, and diffets . While most net- and et - related studies focused on bacterial, viral, and fungal pathogens, little attention was paid to protozoan parasites . As such, the first ever published study on parasite - triggered netosis was published in 2008 by baker et al . 4 years after the discovery of this novel effector mechanism and reported on plasmodium falciparum - triggered net formation . Parasites are mosquito - borne pathogens that cause malaria, a serious public health disease worldwide in the tropic and subtropics . Globally, an estimated 3.3 billion people are at risk of being infected with malaria of whom approximately 1.2 billion are at high risk (> 1 in 1000 chance) of developing malarial disease . The first report on p. falciparum - induced nets referred to p. falciparum - infected children and demonstrated in vivo net - entrapped trophozoite - infected erythrocytes in blood samples . Moreover, baker and colleagues provided first evidence on the involvement of parasite - triggered nets in the pathogenesis of malaria since the high levels of anti - dsdna antibodies were above the predictive levels for autoimmunity . Interestingly, a recent study also indicates the capacity of p. falciparum to inhibit net formation which may be of relevance in immunopathogenesis . Thus, a mosquito - derived salivary protease inhibitor (agaphelin) induced by p. falciparum infection inhibited vertebrate elastase and net formation . Whether this represents a true anti - net mechanism remains to be elucidated . Parasites of the genus eimeria are worldwide of high veterinary and economic importance in livestock, especially in chicken, cattle and small ruminants [95100]. Coccidiosis is a disease with high morbidity in animals of all ages, nonetheless inducing pathogenicity especially in young animals and occasionally causing death of heavily infected animals [99, 102, 103]. Several studies showed that pmn infiltrate intestinal mucosa in response to eimeria infections and are occasionally found in close contact to the parasitic stages in vivo [102, 104107]. Pmn have also been shown to directly interact with e. bovis stages and antigens in vitro, resulting in release of proinflammatory cytokines, chemokines, and inos . Additionally, their phagocytic and oxidative burst activities were enhanced in response to eimeria stages in vitro and in vivo . First indications on eimeria spp . As potent net inducers came from behrendt and colleagues who reported on sporozoites to be entangled by an extracellular network of delicate dna fibres being extruded from pmn in vitro (figure 1(a)). Using extracellular dna measurements and dnase treatments other studies confirmed typical characteristics of nets, such as the colocalization of ne, mpo, and histones in the dna backbone of eimeria - induced net - like structures . Meanwhile, also other pathogenic ruminant eimeria species were shown to induce netosis, such as e. arloingi (figures 2(a) and 2(b)) [24, 27] and e. ninakohlyakimovae (prez, personal communication). Importantly, muoz - caro and colleagues proved nets also to occur in vivo in eimeria - infected gut mucosa . The current data suggest that eimeria - induced netosis is a species- and stage - independent mechanism, since it was induced by sporozoites, merozoites i, or oocysts of different eimeria species [23, 24]. Given that pmn were described to act even in the intestinal lumen via different effector mechanisms [27, 108, 109], it appears likely that interactions of luminal pmn with ingested eimeria oocysts or newly excysted sporozoites may occur [6, 23, 24]. In particular, net - related reactions against oocysts would have a high impact on the ongoing infection since they may hamper proper excystation of infective stages (sporozoites) and, in consequence, dampen the degree of infection at the earliest possible time point in the host . Since e. arloingi sporozoites must egress from the oocyst circumplasm through the micropyle, nets covering this area of the oocyst will have a detrimental effect on proper excystation [6, 24]. The same explanation seems feasible for e. bovis and e. ninakohlyakimovae, regardless of the fact that excystation occurs by rupture of the oocyst walls prior to sporozoites egress from sporocysts . Although all eimeria species tested so far equally induced nets, significant differences in entrapment effectivity were reported amongst different host species, parasite species, and stages . Thus, caprine nets immobilised a high proportion of e. arloingi sporozoites (72%), whilst in the bovine system considerably less parasite stages (e. bovis sporozoites: 43%, b. besnoiti tachyzoites: 34%) were found entrapped in net structures [23, 59]. So far, it remains to be elucidated whether the varying effectivity of nets is based on the pmn origin (goats are generally considered as strong immune responders) or on the parasite species . The molecular basis of eimeria - induced netosis is not entirely understood, so far . Enzyme activity measurements and inhibition studies revealed a key role of nox, ne, and mpo in eimeria - triggered net formation (see table 1) which is in agreement to bacterial, fungal, and parasitic pathogens [14, 25, 59, 65, 75, 110]. Referring to signal cascades, analyses on the grade of phosphorylation revealed a key role of erk1/2 and p38 mapk in sporozoite - exposed bovine pmn . Since respective inhibitor experiments led to decreased parasite - mediated net formation, muoz - caro et al . This finding is in agreement with data on t. gondii - mediated net formation . Referring to ca influx, further inhibition experiments proved e. bovis - mediated netosis as dependent on intracellular ca mobilization, since 2-abp (inhibitor of store - operated ca entry) and bapta - am (binding intracellular ca; muoz - caro, unpublished data) but not egta (inhibitor of ca influx from the extracellular compartment; muoz - caro, unpublished data) significantly blocked parasite - triggered netosis . So far, . Reported on enhanced cd11b surface expression on pmn following e. bovis sporozoite exposure . By antibody - mediated cd11b blockage leading to a significant reduction of parasite - triggered netosis bacteria and fungi netosis was reported as a lethal effector mechanism [31, 42]. However, killing effects of nets were not observed in the case of eimeria spp . So far . Given that eimeria spp . Are obligate intracellular parasites, the main function of nets rather seems to be the extracellular immobilisation of infective stages hampering them from host cell invasion . Accordingly, reduced host cell infections rates were reported for e. bovis and e. arloingi sporozoites when previously exposed to pmn [23, 24]. The same feature was reported for monocyte - preexposed e. bovis sporozoites indicating that this leukocyte cell type also casts ets in response to this parasite stage and that etosis had an impact on parasite invasion . Besides e. bovis, e. arloingi (silva, unpublished data), and e. ninakohlyakimovae (prez et al ., submitted manuscript) were also shown to induce monocytes - derived ets . Furthermore, e. ninakohlyakimovae - induced monocytes - etosis showed a rapid induction of ets release upon viable sporozoites, sporocysts, and oocysts encounters, corroborating a stage - independent process in monocyte - derived etosis . In addition, it was found that caprine monocyte - derived - etosis is nox - dependent . With the upregulation of the genes transcription encoding for il-12 and tnf-, relevant immunoregulatory cytokines with transition properties into the adaptive immunity were also demonstrated in e. ninakohlyakimovae - exposed caprine monocytes (prez et al, submitted manuscript). Since the reduction in infection rates early after infection automatically results in decreased proliferation of the parasite, this indirect et - mediated effect should have a beneficial impact on the outcome of the disease . Despite advantageous properties of ets, their ineffective clearance and/or poor regulation might also bear adverse pathological implications, leading to tissue damage in addition to enhanced local proinflammatory reactions [112, 113]. Toxoplasmosis is caused by the facultative heteroxenous apicomplexan polyxenous protozoan t. gondii representing one of the most common parasitic zoonoses worldwide . Toxoplasma gondii is well known to affect almost all warm - blooded mammals including a wide range of domestic animals, wild mammals, marine mammals, marsupials, and humans [115, 116]. In response to t. gondii infections, pmn are promptly recruited to the site of infection producing a variety of proinflammatory cytokines and chemokines [11, 117]. In addition, pmn are capable of killing t. gondii tachyzoites via phagocytosis [118, 119]. Besides this effector mechanism, human, murine, bovine, and harbour seal (phoca vitulina) pmn additionally perform netosis in reaction to t. gondii tachyzoites (figures 1(c) and 1(d)) [25, 26]. Abi abdallah et al . Showed that netosis was triggered by tachyzoites in a parasite strain - independent fashion as an invasion / phagocytosis - independent process . Interestingly, in the murine toxoplasmosis model, tachyzoites - induced nets were not the result of a random cell lysis, but of a controlled dna release process since lysozyme was still present in pmn after performing netosis [25, 120]. In contrast to eimeria spp ., t. gondii - triggered netosis had modest toxoplasmacidal effects by killing up to 25% of the parasites . Considering the obligate intracellular life style of t. gondii and its enormous proliferative capacity in mammalian host cells, parasite entrapment via nets might be of particular importance in vivo based on its interference with host cell invasion . Consistently, harbour seal pmn - promoted nets significantly hampered host cell invasion of t. gondii tachyzoites in vitro . In vivo evidence of t. gondii - induced netosis was reported in a murine pulmonary infection model, revealing an increase of dsdna contents in the bronchoalveolar lavage fluids of t. gondii - infected mice . As equally reported for several other coccidian parasites [14, 23], t. gondii - induced nets were also proven to be nox-, ne-, mpo-, and ca- (soce) dependent and to be mediated by an erk 1/2-related signalling pathway in pmn (see table 1) [25, 26]. Additionally, in earlier studies, not only the pivotal role of pmn but also the important role of monocytes in toxoplasmosis was clearly demonstrated [121123]; however, their capacity to also induce ets in response to tachyzoite stages was just recently demonstrated . Exposure of harbour seal - derived monocytes to viable t. gondii tachyzoites resulted in a significant induction of monocyte - ets and tachyzoites were firmly entrapped and immobilised within harbour seal monocyte - et structures, hampering parasite replication . Bovine besnoitiosis caused by besnoitia besnoiti is an endemic disease in africa and asia [124126] and considered as emergent in europe . During the acute phase of cattle besnoitiosis, b. besnoiti tachyzoites mainly replicate in host endothelial cells of different organs [28, 128] and, upon release, may be exposed to circulating leukocytes . Besnoitia besnoiti tachyzoites were recently reported as effective inducers of pmn- and monocyte - derived ets (figures 1(e), 1(g), and 1(h)) [28, 59]. In the latter case, a high proportion of pmn was found to be involved in netosis, since up to 76% of encountered pmn were found to participate in netosis leading to the immobilisation of approximately one - third of the parasites . Besnoitia besnoiti - triggered netosis furthermore proved as vitality - independent process that was even induced by soluble parasite molecules (homogenates), though at lower levels . Regarding pmn - derived effector molecules, nox, ne, and mpo proved as essential for efficient b. besnoiti - triggered netosis . Thus, respective enzyme activities were encountered in tachyzoite - exposed pmn and chemical blockage of these enzymes via inhibitors blocked parasite - triggered netosis [28, 59]. In contrast to tachyzoites of t. gondii, entrapped b. besnoiti tachyzoites were neither killed by nets nor ets since their host cell infectivity was entirely restored upon dnase i treatments [28, 59]. Given that b. besnoiti tachyzoites mainly proliferate within endothelial cells during the acute phase, these parasitic stages are released via cell lysis in close proximity to endothelium and are exposed to blood contents, such as leukocytes . Several reports have shown that nets themselves interact with endothelium and may cause endothelial damage or dysfunction [129131]. Since activated endothelial cells may produce a broad panel of immunomodulatory molecules with il-8 or p - selectin having been identified as potent net inducers [129, 132], interactions between infected endothelial cells, b. besnoiti tachyzoites, and nets are quite likely . Recently reported on infection - induced upregulation of endothelial - derived il-8 and p - selectin gene transcription and furthermore presented indications on net formation occurring adjacent to infected endothelium after pmn adhesion assays being performed under physiological flow conditions as the ones present in small vessels . Recent net - related investigations on the closely related cyst - forming apicomplexan protozoa neospora caninum have shown that bovine pmn exposed to viable tachyzoites also result in strong netosis (figure 1(f)). With regard to molecular mechanisms, n. caninum - triggered netosis seems to be p2y2-, nox-, soce-, mpo-, ne-, erk1/2-, p38 mapk-, and pad4-dependent (villagra - blanco et al ., submitted manuscript). Cryptosporidium parvum is an euryxenous apicomplexan parasite with worldwide distribution and high zoonotic potential, mainly affecting young children, immunocompromised humans, and neonatal livestock . Typically, cryptosporidiosis is a water- and food - borne enteric disease that causes diarrhoea, dehydration, weight losses, and abdominal pain and leads to significant economic losses in the livestock industry [133, 134]. After ingestion, sporozoites are released from oocysts into the intestinal lumen and infect small intestine epithelial cells . Recent studies reported on a significant contribution of pmn and macrophages to inflammatory responses in cryptosporidiosis in vivo [136, 137]. Muoz - caro and colleagues reported on nets being cast by both bovine and human pmn in response to c. parvum stages . Parasite - triggered netosis proved stage - independent since it was induced by both sporozoites and oocysts (figure 1(b)). Especially in the latter case parasite stages were occasionally entirely covered with net structures thereby most probably hampering proper sporozoite excystation . Given that pmn were shown as active even within the intestinal lumen [108, 109, 138, 139], these reactions should have a significant impact on ongoing in vivo infection . In vitro infection experiments additionally showed the negative impact of nets on host cell invasion since infection rates were significantly reduced when using pmn - preexposed c. parvum stages . The fact that these reactions were entirely reversible via dnase i treatments rather argued against any cryptosporidicidal effects of nets . The colocalization of ne, histones, and mpo with dna in parasite - mediated extracellular fibres proved classical characteristics of nets and inhibitor experiments emphasized the key role of ne, nox, and mpo in efficient net formation . In agreement with findings on eimeria - induced netosis, inhibition experiments revealed c. parvum - triggered net formation as dependent on intracellular ca release and erk 1/2 and p38 mapk - mediated signalling pathways . Interestingly, c. parvum sporozoite - exposed bovine pmn showed increased gene transcription of proinflammatory molecules, some of which were recently shown as potent net inducers (e.g., il-8 and tnf-) [140, 141] and may have potentiated net reactions . Infections with leishmania spp . Represent a major health problem and according to the who 10% of the human world population is at risk of infection, meaning that approximately 12 million people in 98 countries are infected, and 2 million new cases occur each year [142, 143]. Leishmaniasis is a vector - transmitted zoonosis caused by more than 25 different obligate intracellular protozoan leishmania species [142144]. Particularly pmn have been implicated in the immunopathogenesis of leishmaniasis [145149] and recent studies examined the potential role of nets during the early phase of the disease of different leishmania species . . Showed for the first time that promastigotes of leishmania amazonensis, l. major, and l. chagasi were capable of triggering net formation . Additionally, leishmania - triggered netosis seems not entirely stage - specific, since both promastigotes (l. amazonensis, l. major, l. chagasi, l. donovani, l. mexicana, and l brasiliensis) and amastigotes (l. amazonensis, l. braziliensis) promoted net formation in vitro and in vivo [51, 147, 150152]. More importantly, guimares - costa et al . Provided first indications on possible parasite - specific ligands being responsible for leishmania - mediated netosis . Thus, leishmania - derived lipophosphoglycans (lpg) were suggested as the main trigger of net release since these molecules also induced nets in a purified form . The former authors showed that nets possessed detrimental effects on parasites as net - entrapped l. amazonensis promastigotes exhibited decreased viability . Authors also demonstrated that the extracellular dna and histones found on nets were involved in the parasite inactivation / killing process . The leishmanicidal effects of histones were proven in promastigotes cocultures with purified h2a histones leading to the killing of parasites and by a significant reduction of leishmanicidal effects when cocultured in the presence of anti - histone antibodies . Demonstrated that also the histone h2b could directly and efficiently kill promastigotes of l. amazonensis, l. major, l. braziliensis, and l. mexicana . In case of l. donovani, gabriel et al . Reported netosis as a ros - dependent process which was equally triggered in human and murine pmn (see table 1). However, leishmania - lipophosphoglycan- (lpg-) dependent net induction reported by guimares - costa et al . Was not observed with l. donovani . When using genetically modified l. donovani promastigotes gabriel et al nonetheless, in this infection system, lpg appeared to be involved in the resistance to nets - mediated killing, since the wild type of l. donovani maintained its viability in the presence of nets, whilst mutant parasites lacking lpg were efficiently killed by these extracellular structures . A more recent study revealed that leishmania parasites trigger not only the classical ros - dependent netosis as previously demonstrated but also a ros - independent form, named as early / rapid vital netosis . During this early / rapid leishmania - triggered netosis, in which net formation takes place after 515 min of activation without affecting pmn viability [29, 68], the parasites are also being efficiently entrapped . Regarding net - related evasion strategies of trypanosomatidae parasites, leishmania spp . Seem capable of evading net killing by firstly blocking the oxidative burst activity of pmn or even by resisting microbicidal activity of nets [145, 150]. Moreover, guimares - costa et al . Showed that l. infantum promastigotes express the enzyme 3-nucleotidase / nuclease which was previously described to be involved in parasite nutrition and infection and was proven to be part of the ability of promastigotes to escape net - mediated killing . A recent investigation has shown that a salivary component of the sand fly insect that transmits leishmaniasis may also play a role in the survival of leishmania in the definitive hosts, by modulating their innate immune system . A molecule named lundep from the salivary gland of lutzomyia longipalpis was recently described as an endonuclease with net - destroying properties in humans . In the presence of lundep, measured the ne release from nets as an indicator of net destruction, since ne is normally decorating nets backbone structures and found at low concentrations in culture supernatants, as previously demonstrated . Lundep was responsible for the significant increase of ne concentration in the supernatants when compared to negative controls . In conclusion, these experiments showed degradation of dna scaffold of nets, destroying their functional integrity, and increasing promastigote survival and exacerbating l. major infection . Approximately eight million people are affected by this tropical disease in the americas and an average of 12,000 deaths per year is known to occur due to american trypanosomiasis . It is well known that macrophages, eosinophils, monocytes, and pmn are implicated in the control of early infection [30, 155]. . Demonstrated in vitro that t. cruzi is able to trigger nets in a dose-, time-, and ros - dependent manner . In agreement with reports on eimeria spp . And b. besnoiti [23, 24, 59] but in contrast to observations on t. gondii and leishmania spp . [25, 51], the viability of t. cruzi stages was not affected by nets, but netosis significantly impaired the parasite host cell infectivity . In fact, nets components as ne may affect t. cruzi infectivity, since this enzyme appears to be involved in increased trypanocidal activity and in the reduction of trypomastigote release by prestimulated infected macrophages [30, 156]. Additionally, the authors showed via antibody - mediated blockage that t. cruzi - triggered netosis is a tlr2- and tlr4-dependent process . Moreover, the study showed that not only viable t. cruzi trypomastigote forms but also soluble antigens and killed t. cruzi parasites induced net release in human pmn . In vivo murine studies indicated the relevance of netosis for the outcome of trypanosomiasis since significantly decreased parasites numbers were found in the blood system of those animals which had previously been infected with nets - pretreated parasites . During the last years a vast amount of data on protozoan - mediated etosis was published strengthening the role of this effector mechanism in the defence of parasitic infections . Several in vivo data have now proven the existence and importance of this early host innate effector mechanism . However, there is still a total lack of information on parasite - derived ligands triggering etosis . Taking into account that in most cases et formation is considered as a species- and stage - independent process, rather ubiquitary occurring molecules may represent parasite - derived target molecules of ets . Moreover, recent data revealed that other leukocytes such as monocytes, macrophages, basophils, mast cells, and eosinophils also perform etosis upon pathogen encounter . Furthermore, et - related research mainly focused on the leukocytes aptitude to impact the parasites life cycle, but not on the propensity of parasitic stages to develop counter mechanisms for ets avoidance . While a bunch of data is available on bacterial nucleases or other counter mechanisms, taken together, we call for more parasite - related studies in the exciting field of etosis.

The national tb prevalence survey in eritrea was conducted from february through october 2005 (6). In 40 selected villages, a census (which included information about sex and age) was taken of 875 persons in each village . All persons> 15 years of age were asked to provide a morning and a spot sputum sample . Persons who had 2 positive sputum samples were informed about the test results and referred for treatment . Those who had 1 positive sputum sample were referred to a nearby healthcare facility for further smear examination . If results of smear examination were negative, thoracic radiographs were taken and evaluated by 2 experienced radiologists . The case definition for a sputum smear positive case was at least 2 sputum specimens positive for acid - fast bacilli by ziehl - neelsen staining and microscopy or at least 1 sputum specimen positive for acid - fast bacilli and radiographic abnormalities consistent with active pulmonary tb (classification of the national tuberculosis control program in eritrea). Using the prevalence estimate obtained from the survey and 2 different models, we calculated the cdr for 2004 . In model 1, described by styblo, cdr = (notification rate / prevalence rate) / (0.5 + 0.83 [notification rate / prevalence rate]) (7,8). In model 2, described by dye et al ., cdr = (notification rate / prevalence rate) / ([notification rate / prevalence rate] + 0.5) (9,10). We then compared the calculated cdr with the cdr estimated by the world health organization (who) to evaluate whether comparable conclusions about tb case detection would be obtained . A total of 38,047 persons were included in the prevalence survey . Of those> 15 years of age, 18,152 (94.6%) provided at least 1 sputum sample (figure). The prevalence of new smear - positive tb was estimated at 90/100,000 (95% confidence interval [ci] 35145/100,000) in persons> 15 years of age . In 2005, 44.7% of the eritrean population was <15 years of age (11), which resulted in an overall new smear - positive tb prevalence of 50/100,000 (95% ci 1980/100,000) under the assumption of no cases in persons <15 years of age . Summary of tuberculosis prevalence survey in eritrea, 2005 . In 2004, 17/100,000 new smear - positive cases were reported (2). For eritrea, the cdr provided by who is considerably lower than that calculated from the results of the national tb prevalence survey . Both estimates indicate that eritrea has not reached the 70% target for case detection . However, the who estimate suggests that the program needs to improve case detection by a factor of 5, whereas the survey estimate suggests that case detection needs to be improved by a factor of 1.6 . Two explanations may account for the large difference: 1) the cdr derived from the tb prevalence survey is too high because of an underestimation of the prevalence of smear - positive tb, or 2) the cdr estimate published by who is too low because of an overestimation of the incidence of smear - positive tb . In the national tb prevalence survey, measures were taken to ensure high quality of the results; e.g., training of data collectors, repeat census taking, reexamination of all slides found positive on fluorescence microscopy, and reexamination of a 5% random sample of the negative slides . Persons who had smear - positive tb may have been missed because they did not provide a specimen; however, because only 5% of eligible persons did not provide a specimen, this can explain only a slight underestimation . Furthermore, recorded reasons for not providing a specimen seem to be unrelated to a higher chance of having tb . The quality of the provided specimens may have been suboptimal because instructing and motivating persons to provide a sputum sample is challenging . For diagnosis of tb, microscopic examination of saliva is less sensitive than examination of sputum; however, in 50% of saliva samples from patients with a positive sputum sample, bacilli can be demonstrated (12,13). For 27,647 samples that appeared to be saliva, smear assuming that only 50% were detected, a maximum of 12 smear - positive tb patients may have been undetected . Taking this into account results in a prevalence of 87/100,000 . Using this estimate, model 1 provides a cdr of 30% and model 2 a cdr of 28% the possibility that persons who provided a saliva sample were not able to produce a sputum sample because they did not have pathologic pulmonary changes should also be taken into consideration . Estimation of the incidence of smear - positive tb in eritrea is complicated by the fact that no data from tuberculin or prevalence surveys were available . The only data available for eritrea were reporting data, which experts assessed as being of low quality (14). Use of this limited information will result in an uncertain incidence estimate, which may result in an unreliable cdr . For most countries in africa, little information is available for estimating the prevalence of disease and progress towards the millennium development goals (http://unstats.un.org/unsd/mi/mi_goals.asp, accessed 2006 aug 30). On the basis of case reporting, tb was rightly declared an emergency by african health ministers at the who africa regional committee in maputo in 2005 (15). To be able to fight this emergency, more reliable information about the prevalence of tb in africa is needed . Furthermore, for global tb control, reliable information about the tb epidemic in africa is needed because 28% of the incident smear - positive cases occurred in the who african region in 2004 (2). In conclusion, the example of eritrea shows that a large gap may exist between available estimates of tb prevalence and actual tb prevalence in africa . National tb prevalence surveys in africa would help provide better information on tb prevalence and case detection.

Spermatogenesis is a complex process of development that occurs in most mammals throughout adulthood period . This process happens in seminiferous tubules, lined with epithelial tissue that contains sertoli cells . Sertoli cells are surrounded by a thin septum of peritubular cells that hold keep germ cells (1, 2). The spermatogonia undergo mitotic divisions, meiotic alternations, and morphologically transform to highly developed cells, spermatozoa, with a unique function and structure (3). Such a complex process needs a unique program to control and regulate the involved genes . This gene regulation is accomplished by cell surface molecules which participate in cell to cell and cell to extracellular matrix interactions (4, 5). On the other hand, when sperm is passing through the male and female genital tracts, it gains fertilization ability (capacitation) and adopts maturity characteristics . Maturity is initiated in the epididymis, where sperm communicates with the cell surface molecules of the pathway (6). Therefore, the investigation of stage and/or cell - specific molecular expression in testis or post - testicular maturation system can improve our understanding of germ cell differentiation and sperm formation (7). First identified a 38kda protein (mtex101) in adult mouse testis which is detectable on the surface of spermatocytes and spermatids in testis that it is absent on somatic cells, including sertoli or interstitial cells, like leydig cells (3). There is a temporary expression of mtex101 on oogonia, but the molecule disappears in the mature ovary . In embryonic testis, mtex101 is detected on the prosperm - atogonia (8). Upon puberty, mtex101 mrna is expressed in spermatocytes and in steps 1 - 9 spermatids during spermatogenesis, but not during spermatogonia . The tex101 protein remains on the cell surfaces in steps 10 - 16 spermatids and testicular sperm, but hides from epididymal sperm after passing the caput epididymis (6). The expression of mtex101 homologous protein has been reported in a leukemia cell line and lung cancer in rats and humans, respectively (9, 10). The exact structure and function of mtex101 is not completely understood yet, but it has been speculated that this protein is associated with several transmembrane proteins like ly6k (11) and cellubrevin (12) that transduce the extra - cellular signals to the intracellular molecules . The present study was undertaken to produce recombinant mtex101, which might pave the way for the production of specific antibodies for further structural and functional characterization of the molecule . Total rna was isolated from male gonadal organs (four to 6 week - old mice from pasteur institute of iran). Rna concentration was measured by a biophotometer (eppendorf, hamburg, germany) at 260 nm . One microgram of the total rna was reversetranscribed to cdna by using 200u of molony murine leukemia virus (rtm - mulv) reverse transcriptase enzyme (fermentas, vilnius, lithuania), and 20pmol of random hexamer primers (cybergene, stockholm, sweden). Pcr amplifications for mtex101 transcript was performed in a volume of 25l using 10 - 20ng of testis cdna, forward and reverse primers (10pmol each), specific for mtex101 transcript, 10x pcr buffer (2.5l), dntp mixture (0.2 mm each), 1 mm mgcl2, and 1 unit of taq dna polymerase (roche, mannheim, germany). Pcr reactions were carried out using a thermal cycler (eppendorf mastercycler gradient) as indicated below: a preheating cycle at 95c for 3min, 35 cycles of denaturation at 95c for 30sec, annealing at 60c for 30sec, extension at 72c for 60sec, and finally a 7-min cycle at 72c . The forward and reverse mtex101 orf primers used (forward: 5ccg aat tca tgg gag cct gcc gca tcc ag 3, reverse: 5 agg gaa gtg ggt gag ggg gga gca gag cgg ccg c 3. ), contained ecori and noti restriction enzyme (re) sites, respectively . The pcr products were resolved by ethidium bromide - stained 1.5% agarose gel electrophoresis, which revealed a single distinct band at 750bp (figure 1). The 750bp band was purified using qia quick gel extraction kit (qiagen, germantown, md, usa). Pcr optimization on mouse testis cdna by mgcl2 gradients . 1 - 4: mgcl2 concentrations from 1 to 4 mm, respectively, 5: 1 kb dna ladder, 6: negative control (no dna). The pgem - t easy vector (promega, madison, wi, usa) was used for the cloning of the purified pcr product . Ligation reac tion was set up by 50ng of pgem - t easy vector (promega), three units of t4 dna ligase, 75ng of mtex101 purified fragment, and 6l of rapid 2x ligation buffer (promega). Competent cells of e. coli jm109 strain were used for transformation through heat shock method (17). The transformed bacteria were left for 1 hr in lb broth at 37c for recovery, and later 100l of the transformation culture was plated onto an ampicillin (100mg / ml) (sigma, louis, mo, usa), iptg (sigma) (0.5 mm) and x - gal (sigma), (80g / ml) containing lb agar plate and was cultured for 16hrs at 37c . The recombinant plasmids were isolated from confirmed colonies by miniperp kit (qiagen) and then digested by ecori and noti restriction enzymes . Plasmid dna (800ng) was used in 25l of the total volume, including noti (15units), (invitrogen, carlsbad, ca, usa), ecori (15 units), (invitrogen), and 2.5l of 10 x reaction buffer 3 (invitrogen) and incubated for 1.5 hr at 37c . The mtex101 fragment in pgem - t easy was extracted form agarose gel and then subcloned into a pet-28a (+) expression vector (merck, darmstadt, germany) and was digested by the same restriction enzymes above . Ligation reaction was performed by 125ng of digested pet-28a (+), 0.5pmol of mtex101 purified fragments, 2l of rapid 2x ligation buffer (pro - mega), and 3 units of t4 dna ligase (promega) in 10l of the total volume and it was incubated overnight at 4c . Ligation products were used for transformation of e. coli jm109 strain by heat shock method (17). After a recovery time of 1 hour in lb broth, the transformed bacteria were cultured on lb agar and kanamycin (50g / ml) (sigma, usa) containing plates for 16 hrs at 37c . The obtained colonies were screened by colony pcr and one of them was confirmed by dna sequencing . Recombinant plasmid was purified and was then used for transformation of bl21 (de3), which is a protease deficient strain of e. coli . A single transformed colony was inoculated into 50ml of lb broth, including kanamycin (50g / ml) (sigma, usa) and untransformed bl21 (de3) bacteria were cultured into 50 ml of lb medium . Incubation was performed under shaking at 37c until the optical density (od) at 600 nm reached 0.6 . Iptg (sigma) was added to the remainder with the final concentration of 1 mm and incubation continued for 2 - 3 more hours . Cell lysates were prepared by sonication of pellets in pbs buffer including 1% protease inhibitor (roche). Protein solutions were obtained from bacteria by boiling them in a sample buffer that contained tris - hcl (ph=6.8, 0.5 m), sds (10% w / v), glycerol (50% v / v), and bromo phenol blue (0.5% w / v), for 5min . Samples (50l of each) were separated by sds - page (8%) (bio - rad, hercules, ca, usa) under non - reducing conditions . The protein constituents were then electro - phoretically blotted onto a polyvinylidene difluoride (pvdf) membrane (millipore, billerica, ma, usa), as described by towbin et al . (the membrane was blocked in 5% skim milk / 0.1% tween 20 in pbs (ph 7.4), and then the reactivity of the transferred protein(s) with 1g / ml of anti mtex101 pab (avicenna research institute, iran) was assessed using sheep anti rabbit ig hrp (avicenna research institute, iran). Finally, the membranes were visualized using an ecl system (ge healthcare, biotech, bucking - hamshire, uk). Total rna was isolated from male gonadal organs (four to 6 week - old mice from pasteur institute of iran). Rna concentration was measured by a biophotometer (eppendorf, hamburg, germany) at 260 nm . One microgram of the total rna was reversetranscribed to cdna by using 200u of molony murine leukemia virus (rtm - mulv) reverse transcriptase enzyme (fermentas, vilnius, lithuania), and 20pmol of random hexamer primers (cybergene, stockholm, sweden). Pcr amplifications for mtex101 transcript was performed in a volume of 25l using 10 - 20ng of testis cdna, forward and reverse primers (10pmol each), specific for mtex101 transcript, 10x pcr buffer (2.5l), dntp mixture (0.2 mm each), 1 mm mgcl2, and 1 unit of taq dna polymerase (roche, mannheim, germany). Pcr reactions were carried out using a thermal cycler (eppendorf mastercycler gradient) as indicated below: a preheating cycle at 95c for 3min, 35 cycles of denaturation at 95c for 30sec, annealing at 60c for 30sec, extension at 72c for 60sec, and finally a 7-min cycle at 72c . The forward and reverse mtex101 orf primers used (forward: 5ccg aat tca tgg gag cct gcc gca tcc ag 3, reverse: 5 agg gaa gtg ggt gag ggg gga gca gag cgg ccg c 3. ), contained ecori and noti restriction enzyme (re) sites, respectively . The pcr products were resolved by ethidium bromide - stained 1.5% agarose gel electrophoresis, which revealed a single distinct band at 750bp (figure 1). The 750bp band was purified using qia quick gel extraction kit (qiagen, germantown, md, usa). Pcr optimization on mouse testis cdna by mgcl2 gradients . 1 - 4: mgcl2 concentrations from 1 to 4 mm, respectively, 5: 1 kb dna ladder, 6: negative control (no dna). The pgem - t easy vector (promega, madison, wi, usa) was used for the cloning of the purified pcr product . Ligation reac tion was set up by 50ng of pgem - t easy vector (promega), three units of t4 dna ligase, 75ng of mtex101 purified fragment, and 6l of rapid 2x ligation buffer (promega). Competent cells of e. coli jm109 strain were used for transformation through heat shock method (17). The transformed bacteria were left for 1 hr in lb broth at 37c for recovery, and later 100l of the transformation culture was plated onto an ampicillin (100mg / ml) (sigma, louis, mo, usa), iptg (sigma) (0.5 mm) and x - gal (sigma), (80g / ml) containing lb agar plate and was cultured for 16hrs at 37c . The recombinant plasmids were isolated from confirmed colonies by miniperp kit (qiagen) and then digested by ecori and noti restriction enzymes . Plasmid dna (800ng) was used in 25l of the total volume, including noti (15units), (invitrogen, carlsbad, ca, usa), ecori (15 units), (invitrogen), and 2.5l of 10 x reaction buffer 3 (invitrogen) and incubated for 1.5 hr at 37c . The mtex101 fragment in pgem - t easy was extracted form agarose gel and then subcloned into a pet-28a (+) expression vector (merck, darmstadt, germany) and was digested by the same restriction enzymes above . Ligation reaction was performed by 125ng of digested pet-28a (+), 0.5pmol of mtex101 purified fragments, 2l of rapid 2x ligation buffer (pro - mega), and 3 units of t4 dna ligase (promega) in 10l of the total volume and it was incubated overnight at 4c . Ligation products were used for transformation of e. coli jm109 strain by heat shock method (17). After a recovery time of 1 hour in lb broth, the transformed bacteria were cultured on lb agar and kanamycin (50g / ml) (sigma, usa) containing plates for 16 hrs at 37c . The obtained colonies were screened by colony pcr and one of them was confirmed by dna sequencing . Recombinant plasmid was purified and was then used for transformation of bl21 (de3), which is a protease deficient strain of e. coli . A single transformed colony was inoculated into 50ml of lb broth, including kanamycin (50g / ml) (sigma, usa) and untransformed bl21 (de3) bacteria were cultured into 50 ml of lb medium . Incubation was performed under shaking at 37c until the optical density (od) at 600 nm reached 0.6 . Iptg (sigma) was added to the remainder with the final concentration of 1 mm and incubation continued for 2 - 3 more hours . Cell lysates were prepared by sonication of pellets in pbs buffer including 1% protease inhibitor (roche). Protein solutions were obtained from bacteria by boiling them in a sample buffer that contained tris - hcl (ph=6.8, 0.5 m), sds (10% w / v), glycerol (50% v / v), and bromo phenol blue (0.5% w / v), for 5min . Samples (50l of each) were separated by sds - page (8%) (bio - rad, hercules, ca, usa) under non - reducing conditions . The protein constituents were then electro - phoretically blotted onto a polyvinylidene difluoride (pvdf) membrane (millipore, billerica, ma, usa), as described by towbin et al . (13). The membrane was blocked in 5% skim milk / 0.1% tween 20 in pbs (ph 7.4), and then the reactivity of the transferred protein(s) with 1g / ml of anti mtex101 pab (avicenna research institute, iran) was assessed using sheep anti rabbit ig hrp (avicenna research institute, iran). Finally, the membranes were visualized using an ecl system (ge healthcare, biotech, bucking - hamshire, uk). After cloning mtex101 fragments in pgem - t easy vector, several white colonies with probable target fragment inclusion were screened by colony pcr (figure 2). A right - sized pcr product and a pet-28a(+) vector were cut by noti and ecori restriction enzymes to obtain the required fragments for the next step (figures 3 and 4). Coloy pcr on transformed jm109 clones by pgem - t easy vector carrying mtex101 gene . 1 - 5: 5 selected white colonies, 6: negative control (blue colony), 7: positive control (pcr product on testis cdna), 8: dna ladder viii (roche). Double digestion of pgem - t easy vector containing mtex101 fragment with ecori and noti restriction enzymes . 1: digested vector with mtex101 750bp insert cut out of the vector, 2: dna ladder viii . Double digestion of pet-28a (+) expression vector with restriction enzymes . 1: digested pet-28a (+) with ecori and noti, 2: uncut pet-28a (+), 3: 1 kb dna ladder . E. coli j m109 strains were transformed using recombinant pet-28a(+) containing mtex101 fragment and the undertaken transformation was verified by colony pcr (figure 5). One of the confirmed colonies (clone 2 in figure 5) was picked for detailed analysis by dna sequencing . Alignment of dna sequencing results with mtex101 orf by genbee site (14) confirmed the cloned sequence . The verified fragment was used for the next round of transformation using e - coli bl21 (de3) strain . Colony pcr on transformed jm109 clones by pet-28a (+) vector containing mtex101 fragment . 1 and 2 represent two selected colonies, 3: dna ladder viii, 4: negative control (no dna), 5: positive control (pgem - t easy vector containing mtex101 fragment). Western blot analysis using anti - mte x 101 peptide polyclonal antibody revealed the correct size of mtex101 recombinant protein production (27kda) in bl-21 (de3) bacteria (figure 6). Western blot analysis of production of the recombinant mtex101 in bl21 (de3) bacteria . 1: bl21 (de3) containing mtex101 gene after induction, 2: bl21 (de3) containing mtex101 gene before induction, 3: untransfected bl21 (de3), 4: protein marker (see blue - invitrogen). After cloning mtex101 fragments in pgem - t easy vector, several white colonies with probable target fragment inclusion were screened by colony pcr (figure 2). A right - sized pcr product and a pet-28a(+) vector were cut by noti and ecori restriction enzymes to obtain the required fragments for the next step (figures 3 and 4). Coloy pcr on transformed jm109 clones by pgem - t easy vector carrying mtex101 gene . 1 - 5: 5 selected white colonies, 6: negative control (blue colony), 7: positive control (pcr product on testis cdna), 8: dna ladder viii (roche). Double digestion of pgem - t easy vector containing mtex101 fragment with ecori and noti restriction enzymes . 1: digested vector with mtex101 750bp insert cut out of the vector, 2: dna ladder viii . Double digestion of pet-28a (+) expression vector with restriction enzymes . 1: digested pet-28a (+) with ecori and noti, 2: uncut pet-28a (+), 3: 1 kb dna ladder . E. coli j m109 strains were transformed using recombinant pet-28a(+) containing mtex101 fragment and the undertaken transformation was verified by colony pcr (figure 5). One of the confirmed colonies (clone 2 in figure 5) was picked for detailed analysis by dna sequencing . Alignment of dna sequencing results with mtex101 orf by genbee site (14) confirmed the cloned sequence . The verified fragment was used for the next round of transformation using e - coli bl21 (de3) strain . Colony pcr on transformed jm109 clones by pet-28a (+) vector containing mtex101 fragment . 1 and 2 represent two selected colonies, 3: dna ladder viii, 4: negative control (no dna), 5: positive control (pgem - t easy vector containing mtex101 fragment). Western blot analysis using anti - mte x 101 peptide polyclonal antibody revealed the correct size of mtex101 recombinant protein production (27kda) in bl-21 (de3) bacteria (figure 6). Western blot analysis of production of the recombinant mtex101 in bl21 (de3) bacteria . 1: bl21 (de3) containing mtex101 gene after induction, 2: bl21 (de3) containing mtex101 gene before induction, 3: untransfected bl21 (de3), 4: protein marker (see blue - invitrogen). The researchers immunized female mice with 8-week old testis lysates and obtained 12 monoclonal antibody producing clones (tes101 - 112). Immunohisto - chemistry analyses revealed that only tes101 was able to recognize a novel testicular protein as determined by searching the expressed sequence tag (est) database (3). All studies on mtex101 protein are performed solely by tes101 antibody and presently, no commercial antibodies are available to carry out research on this protein . In this study, we produced mtex101 recombinant protein that is useful for antibody development for further investigations . Mtex101 mrna contains 750 bases, which encodes 250 amino acids . After the removal of the putative 25-amino acid signal peptide at n - terminus reported a 38kda band by western blot analysis under non - reducing conditions and no 24 kda band was observed (3). This large difference in the molecular mass was speculated to be due to glycosylation of the peptide and it was proved to be true by jin et al . Glycosylation is known as the prime cause of post - translational modifications (ptm) in proteins (16). The asn - x - ser / thr is the basic sequence for n - linked glycosylation, however, the secondary structure of the protein can affect the final addition . No consensus sequence has been established for o - linked glycosylation yet (17). Jin et al, found four putative sites for n - glyco - sylation and several possible sites for o - glyco - sylation in mtex101 amino acid sequence (15). In fact, mtex101 is a highly glycosylated protein and it has been clarified that most of the oligosaccharide chains on this peptide are n - linked carbohydrates (12). De - n - glycosylation of mtex101 created an 20kda band which is close to or even smaller than estimated molecular weight of mtex101 amino acid backbone (15), confirming glycosylation as the cause for the higher mass of native mtex101 . The cellular role of glycoprotein sugar components can be investigated via several methods, like protein production in a host lacking oligo - saccharide addition system, such as prokaryotes (18). We succeeded to produce mtex101 recombinant protein in a prokaryotic system, which may be useful in clarifying the role of sugar components in protein structures . However, proteins which are produced by this method may vary in their structures and functions (19). Western blot analysis showed that this protein had a molecular mass of approximately 27kda under non - reducing conditions that is concordant with the various post - translational modification processes in prokaryotic systems as compared to those in eukaryotes . The size of the recombinant protein (27kda) is in conformity with the backbone protein (24kda) and a 3kda peptide derived from the digested vector by noti and ecori restriction enzymes . In this study, we successfully cloned mtex101 into a his - tagged expression vector (pet-28a(+)), followed by an efficient production of the relevant recombinant protein . This protein can be used in antibody production to find out the role of mtex101 in spermatogenesis, egg fertilization and further investigation about the critical role of glycosylation in the function of this protein.

Most malignancies of the upper aerodigestive tract (figure 1), comprising the naso-, oro-, hypo-, and laryngopharynx, are squamous cell carcinomas . Head and neck squamous cell carcinomas (hnsccs) are the primary tumor type in head and neck cancer (hnc), characterized by local tumor aggressiveness, high rate of early recurrences, metastasis, and development of second primary tumors, which are the major cause of morbidity and mortality in hnscc (details in [14]). More than 90% of hnc cases are induced by chronic exposure to carcinogens enclosed in all forms of tobacco, synergized by heavy alcohol consumptions and poor diet (see [5, 6]). It is estimated that about 5%10% of suspicious lesions arising in the mucous membranes of the mouth, pharynx, and larynx undergo malignant transformation . Cure rates of early disease (stage i and ii) range between 70% and 80%, and chemoprevention strategies seem promising to control potentially malignant oral lesions (reviewed in [13]). However, long - term survival rates, especially for advanced hnc, have not improved significantly over the last decades . Despite modern therapeutic strategies and sophisticated surgical management of the tumor, the estimated five - year survival rate for advanced disease (30%40%) remains poor ([13] and references therein). Currently, rational therapeutic strategies targeting growth factor receptors by specific antibodies or kinase inhibitors have gained increasing clinical relevance in particular for the treatment of locally advanced cancer with the intent of preserving speech and swallowing (see [13]). Thus, developing new therapeutic strategies and defining novel target proteins for the treatment of advanced hnc is of particular importance . In this respect, nuclear receptors (nrs) are transcription factors implicated in cancer development and are recently attracting major interest as therapeutic targets (see [7, 8]). As nrs modulate cell proliferation, apoptosis, invasion, and migration, clearly representing hallmarks of cancer cells, several highly successful cancer drugs target this receptor family [811]. Since several nrs have been shown to be expressed also in head and neck cancer cells, nrs are most likely also contributing to hnscc development and progression [12, 13]. Nrs belong to a large superfamily of transcription factors and based on sequence comparison are currently classified into seven subfamilies (table 1). These transcription factors are able to modulate transcription of a variety of target genes by several distinct mechanisms, including both transcriptional activation and repression [7, 8, 14, 15]. Transcriptional regulation can either be ligand - dependent or -independent, genomic or nongenomic, allowing nrs to mediate gene repression or its release, gene activation, or gene trans - repression (details in [7, 8, 16]). In particular, the large group of so - called orphan nuclear receptors, for which natural ligands are still unknown, do not exist at all (true orphans), or have only recently been identified (adopted orphans) is adding additional complexity to the field (table 1) ([8, 17], and references within). In contrast to cell surface growth factor receptors, such as the epidermal growth factor receptor (egfr), which activate genetic programs through complex intracellular signaling cascades, nrs are able to directly bind to specific dna - sequences, so - called hormone response elements (hres). Thus, nrs are composed of an n - terminal regulatory domain (activation function 1 = af1), followed by a dna - binding domain (dbd), a ligand - binding domain (lbd), and another c - terminal regulatory domain (activation function 2 = af2) (figure 2) [7, 8]. Despite their conserved structural organization, nevertheless, two major modes of nr action can be assigned, depending on their intracellular steady - state localization in the absence of ligands (figure 3). One group of nrs is confined to the cytoplasm within multiprotein - complexes in the absence of ligand . Upon ligand binding, they actively enter the nucleus and bind to hres as homo- or heterodimers (figure 4, details in [7, 8]). Other nrs already reside in the nucleus in a complex with corepressor proteins, while ligand binding triggers corepressor dissociation allowing the recruitment of coactivators [18, 19]. However, in order to fulfill multiple biological tasks minor to major deviations from these two modes of nr action exist [7, 8]. Nrs are not only implicated in a broad spectrum of physiological processes but are associated with many human diseases including metabolic and cardiovascular disorders as well as cancer . Beside their proven clinical relevance for hormone regulated malignancies, there is rather limited information on their pathophysiological role as well as their prognostic and therapeutic potential for head and neck cancer [7, 8, 12, 2022]. Most studies were investigating members of two classes of the nr superfamily, the thyroid hormone receptor - like and the estrogen receptor - like receptors (table 1). Thus, we will focus on relevant members of these subfamilies, summarize their potential diagnostic / prognostic value, and discuss their therapeutic potential . Within the thyroid hormone receptor - like receptor subfamily, the peroxisome proliferator - activated receptors (ppars) show the highest disease relevance for hnscc . To date, three isoforms of the ppar (, /, and) have been identified, all able to form heterodimers with retinoid x receptors (rxrs) (see [23, 24]). Ppars are expressed in different cell types and activate the transcription of several genes involved in a variety of biological processes, including lipid metabolism and insulin sensitivity (see [23, 24]). Furthermore, a role in limiting inflammation has also been reported [24, 25]. As tumor cell metabolism and inflammation appear to be critical for tumorigenesis and clinical outcome, nrs may thus directly or / and indirectly modulate malignancies [26, 27]. As such, ppar is overexpressed in many epithelial malignancies [22, 28, 29] including oral squamous cell carcinoma . In the absence of ligand, ppars are complexed with corepressor proteins, thus acting as transcriptional repressors . Ligand binding induces conformational changes facilitating heterodimerization with rxr, thus leading to the attraction of transcriptional coactivators (figures 3 and 4) (see [19, 24]). Natural and synthetic ligands for ppars include lipophilic molecules such as fatty acids and eicosanoids as well as thiazolidinedione (tzd) drugs and derivates thereof (overview in [7, 24, 31]). Ppar ligands seem to exert their effects in a dosage - dependent manner, although the detailed mechanism is currently not yet resolved . The postulated cancer modulating mechanisms are diverse, including effects on wnt signaling, inhibition of nfb, as well as the modulation of cell cycle regulators and pro- and antiapoptotic proteins, which have been linked with head and neck cancer (see [4, 23]). Clinical aspects of peroxisome proliferator - activated receptors in hnsccin hnscc, overexpression on the protein level has been convincingly demonstrated for ppar and ppar [12, 30, 33]. Agonist binding to ppar can induce cell differentiation, growth arrest, and apoptosis of cancer cells . Additionally, such activating ppar ligands were shown to exert antiproliferative on human colon and breast cancers (details in [23, 24]) and were also suggested as potential chemopreventive agents for oral carcinogenesis [12, 35, 36]. Of note, since at least 1.6 million patients take antidiabetic drugs that function as ppar ligands, epidemiological data on their long - term effects on tumor prevention would therefore be of value to rationally design cancer chemoprevention trials . Paradoxically, not only ppar agonists are considered as potential therapeutic agents in cancer therapy but also antagonists were studied in this respect . Ppar inhibition was shown to induce apoptosis and anoikis and inhibit tumor cell invasion in squamous cell carcinomas . Moreover, the results of several studies indicated that the growth - inhibiting activity of ppar ligands in oscc may be ppar independent . Others showed that the observed effects were strongly dependent on ppar-expression [12, 38] as well as on the type and concentration of the agonist . In the majority of oscc cases, ppar mrna could be detected by rt - pcr . By immunohistochemical analysis of primary tumors, ppar was often found in low - grade tumors, especially in tumor endothelium, and a favorable impact of ppar expression on relapse - free survival of the patients could be demonstrated . The beneficial effects of ppar ligands on malignancies were tested in several clinical trials, but outcomes proved to be highly diverse . Some trials revealed 40% partial response rates, whereas others could not show any significant beneficial effect [41, 42]. Moreover, one may speculate that the tumor modulating effects of ppar ligands are mediated indirectly by affecting the tumor microenvironment, such as cancer - associated fibroblasts or tumor endothelial cells . In fact, ppar ligands have been shown to affect endothelial cell proliferation and migration and hence to regulate angiogenesis . Also hypoxia - induced angiogenesis appears to be affected by ppar ligands in cancer therapy, even if the precise mechanisms still remain unclear . As angiogenesis is a crucial aspect for tumor development, therapy resistance and metastasis and inhibition of angiogenesis may hence have contributed to the clinical benefit observed.in sum, ppar ligands appear to be of clinical benefit for the treatment of head and neck cancer, in particular for oscc . Nevertheless, a more detailed molecular knowledge on ppar biology is clearly required . Increasing knowledge about the mode of action, specificity, and dosage - dependence of ppar agonistic and antagonistic ligands will hopefully allow a better modeling of ppar receptor function and thus lead to a more effective design of combinatorial application schemes for cancer treatment and cancer prevention in the future . In hnscc, overexpression on the protein level has been convincingly demonstrated for ppar and ppar [12, 30, 33]. Agonist binding to ppar can induce cell differentiation, growth arrest, and apoptosis of cancer cells . Additionally, such activating ppar ligands were shown to exert antiproliferative on human colon and breast cancers (details in [23, 24]) and were also suggested as potential chemopreventive agents for oral carcinogenesis [12, 35, 36]. Of note, since at least 1.6 million patients take antidiabetic drugs that function as ppar ligands, epidemiological data on their long - term effects on tumor prevention would therefore be of value to rationally design cancer chemoprevention trials . Paradoxically, not only ppar agonists are considered as potential therapeutic agents in cancer therapy but also antagonists were studied in this respect . Ppar inhibition was shown to induce apoptosis and anoikis and inhibit tumor cell invasion in squamous cell carcinomas . Moreover, the results of several studies indicated that the growth - inhibiting activity of ppar ligands in oscc may be ppar independent . Others showed that the observed effects were strongly dependent on ppar-expression [12, 38] as well as on the type and concentration of the agonist . In the majority of oscc cases, ppar mrna could be detected by rt - pcr . By immunohistochemical analysis of primary tumors, ppar was often found in low - grade tumors, especially in tumor endothelium, and a favorable impact of ppar expression on relapse - free survival of the patients could be demonstrated . The beneficial effects of ppar ligands on malignancies were tested in several clinical trials, but outcomes proved to be highly diverse . Some trials revealed 40% partial response rates, whereas others could not show any significant beneficial effect [41, 42]. Moreover, one may speculate that the tumor modulating effects of ppar ligands are mediated indirectly by affecting the tumor microenvironment, such as cancer - associated fibroblasts or tumor endothelial cells . In fact, ppar ligands have been shown to affect endothelial cell proliferation and migration and hence to regulate angiogenesis . Also hypoxia - induced angiogenesis appears to be affected by ppar ligands in cancer therapy, even if the precise mechanisms still remain unclear . As angiogenesis is a crucial aspect for tumor development, therapy resistance and metastasis and inhibition of angiogenesis may hence have contributed to the clinical benefit observed . In sum, ppar ligands appear to be of clinical benefit for the treatment of head and neck cancer, in particular for oscc . Increasing knowledge about the mode of action, specificity, and dosage - dependence of ppar agonistic and antagonistic ligands will hopefully allow a better modeling of ppar receptor function and thus lead to a more effective design of combinatorial application schemes for cancer treatment and cancer prevention in the future . Another group of thyroid hormone receptor - like receptors implicated in hnscc is the retinoid acid receptors (rars). Rars are characterized by their activation via vitamin a derivatives . Upon activation, rars are able to heterodimerize with retinoid x receptors (rxr) and to bind to specific hormone response elements (hres), thereby modulating transcription of target genes (figures 3 and 4) [8, 26, 46]. To date, a variety of coactivator- and corepressor - proteins have been identified, allowing the fine - tuning of target gene transcription, ranging from repression to full activation . However, the molecular details are just beginning to be uncovered [8, 26, 46]. Rar activation often leads to differentiation, cell - cycle arrest, or apoptosis, culminating in the inhibiting of tumor growth . Hence, its ligand retinoid acid (ra) or derivates thereof are currently tested as therapeutic agents in several cancer types (table 2). Paradoxically, in some malignancies ra rather promotes cell survival, which may be due to the ability of ra to also activate ppars, and as a consequence expression of prosurvival genes is induced . Could also show that the channeling of ra between these two nuclear receptor heterodimers is mediated by the cytoplasmic ra transporters crabp2 and fabp5 and thus is strongly depending on the fabp5/crabp2 ratio . Thus, the channeling of ra to different receptor heterodimers appears to be crucial for the regulation of cell - proliferation, positively or negatively affecting tumor growth . Interestingly, both proteins were found differentially expressed in metastatic and hpv - associated hnscc, but their biological and clinical effects remain to be investigated [51, 52]. An additional way of biological regulation is epigenetic modulation playing an important role in cancer development (reviewed in). Gene silencing caused by aberrant hypermethylation of cpg islands has not only been detected in promoter regions of several tumor suppressor genes, but several studies show hypermethylation of the rar promoter in colon, breast, and lung cancers [54, 55]. In head and neck carcinogenesis, hypermethylation of the rar promoter was found to be indeed associated with rar downregulation and hence appears to be biologically relevant . Clinical aspects of retinoid acid receptors in hnsccas outlined above, a rationale for the use of retinoids in chemoprevention and cancer therapy was provided experimentally by different cellular and animal models . Moreover, this strategy was supported by epidemiological data as well as by clinical trial outcomes [26, 5961]. Several clinical chemoprevention trials including patients with increased risk for developing cancer have shown that treatment with retinoids resulted in the suppression of precancerous lesions (see [26, 60]). Also, certain retinoids inhibited the development of second primary tumors in patients who had been previously treated for an early - stage cancer but remained at high risk to relapse ([26, 60] and references within). However, other studies using isotretinoin or other retinoids (e.g., retinyl palmitate) did not observe any benefit in second primary tumor development, recurrence, or mortality of hnscc or lung cancer [26, 62]. Current trials (table 2) are therefore aiming to resolve these controversies by recruiting appropriate study populations as well as by the use of novel drugs and improved treatment protocols.reduced rar mrna levels have been observed not only in several malignant tumors ([26, 56] and references therein) but also in premalignant oral lesions (and references within). Thus, most of the studies demonstrating rar downregulation were based on in situ hybridization and could therefore only show a decrease in the amount of mrna . Were the first to demonstrate decreased expression of the rxr and rar// on protein level correlating with different stages of oscc development and progression . The molecular mechanism leading to downregulation or loss of rar is poorly understood, but it was suggested that expression of rar could depend on the intracellular level of retinoids . Several studies demonstrated a decrease in the amount of rar during vitamin a deficiency as well as its upregulation by ra . Additionally, there is evidence that retinoic acid induces the expression of rar mrna in certain cell lines, but not in the malignant counterparts of these cells . Thus, transformed cells may have developed an aberrant response to retinoic acid due to the deregulated expression of coactivator / repressor proteins . Ralhan et al . Found a significant association between the increase in rar mrna levels and clinical responses of premalignant oral lesions to isotretinoin [65, 66]. Hence, rar indeed seems to contribute to the suppression of the premalignant phenotype and malignancy and may be causally linked to the clinical outcome in chemoprevention trials with retinoids [26, 67]. If so, rar may indeed serve as a useful diagnostic marker in retinoid trials (table 2) for the prevention of oral carcinogenesis . Rar modulation by its agonist ligand all - trans retinoic acid (atra) represents a successful example of how targeting of an nr contributes to an impressive clinical benefit in liquid tumors (and references therein). Lessons learned from these studies clearly show that the therapeutic benefit could be further enhanced by combining atra with chromatin modulating agents, such as histone deacetylase inhibitors . Nevertheless, the design of receptor specific drugs as well as an in depth understanding of the molecular regulation of rar biology is required in order to fully exploit its therapeutic benefit and minimize potential side - effects in the area of head and neck cancer [7, 26]. As outlined above, a rationale for the use of retinoids in chemoprevention and cancer therapy was provided experimentally by different cellular and animal models . Moreover, this strategy was supported by epidemiological data as well as by clinical trial outcomes [26, 5961]. Several clinical chemoprevention trials including patients with increased risk for developing cancer have shown that treatment with retinoids resulted in the suppression of precancerous lesions (see [26, 60]). Also, certain retinoids inhibited the development of second primary tumors in patients who had been previously treated for an early - stage cancer but remained at high risk to relapse ([26, 60] and references within). However, other studies using isotretinoin or other retinoids (e.g., retinyl palmitate) did not observe any benefit in second primary tumor development, recurrence, or mortality of hnscc or lung cancer [26, 62]. Current trials (table 2) are therefore aiming to resolve these controversies by recruiting appropriate study populations as well as by the use of novel drugs and improved treatment protocols . Reduced rar mrna levels have been observed not only in several malignant tumors ([26, 56] and references therein) but also in premalignant oral lesions (and references within). Unfortunately, until recently no antibodies convincingly detecting rar were available . Thus, most of the studies demonstrating rar downregulation were based on in situ hybridization and could therefore only show a decrease in the amount of mrna . Were the first to demonstrate decreased expression of the rxr and rar// on protein level correlating with different stages of oscc development and progression . The molecular mechanism leading to downregulation or loss of rar is poorly understood, but it was suggested that expression of rar could depend on the intracellular level of retinoids . Several studies demonstrated a decrease in the amount of rar during vitamin a deficiency as well as its upregulation by ra . Additionally, there is evidence that retinoic acid induces the expression of rar mrna in certain cell lines, but not in the malignant counterparts of these cells . Thus, transformed cells may have developed an aberrant response to retinoic acid due to the deregulated expression of coactivator / repressor proteins . Found a significant association between the increase in rar mrna levels and clinical responses of premalignant oral lesions to isotretinoin [65, 66]. Hence, rar indeed seems to contribute to the suppression of the premalignant phenotype and malignancy and may be causally linked to the clinical outcome in chemoprevention trials with retinoids [26, 67]. If so, rar may indeed serve as a useful diagnostic marker in retinoid trials (table 2) for the prevention of oral carcinogenesis . Rar modulation by its agonist ligand all - trans retinoic acid (atra) represents a successful example of how targeting of an nr contributes to an impressive clinical benefit in liquid tumors (and references therein). Lessons learned from these studies clearly show that the therapeutic benefit could be further enhanced by combining atra with chromatin modulating agents, such as histone deacetylase inhibitors . Nevertheless, the design of receptor specific drugs as well as an in depth understanding of the molecular regulation of rar biology is required in order to fully exploit its therapeutic benefit and minimize potential side - effects in the area of head and neck cancer [7, 26]. This subfamily is composed of the estrogen receptors (er and er), the estrogen - related receptor, and the 3-ketosteroid receptors . Besides the estrogen receptors themselves, many of the genes regulated by the er / estrogen - axis are critical for cell proliferation, inhibition of apoptosis, stimulation of invasion and metastasis, as well as for the promotion of angiogenesis (see [10, 11] and references within). Since these processes clearly state hallmarks of cancer cells, it is well accepted that ers are implicated in various cancer types [9, 21]. Sex hormone receptors are expressed not only in sexual organs but, amongst others, also in the vascular epithelium, the lung epithelium, and the larynx . The expression of sex hormone receptors could also be demonstrated in hnscc by several studies [12, 13]. Both er isoforms as well as the progesterone receptor (pr) were detectable in cancer cells of the oral cavity, the salivary gland, and in laryngeal / hypopharyngeal cancers, whereas the tumor stroma was mostly negative [12, 13]. Expression of er inversely correlated with that of er in esophageal carcinoma, and a correlation of er levels with tumor dedifferentiation and staging was suggested [73, 74]. Clinical aspects of estrogen receptors in hnsccconsidering the impressive benefit of endocrine therapy in breast cancer, targeting sex steroid hormone receptor as a potential therapeutic strategy is also discussed for hnscc [12, 75]. Currently, two main strategies are pursued in endocrine therapy of er - positive tumors . One is based on steroidal antiestrogens like tamoxifen, which bind to the er, block its function, and ultimately induce receptor degradation [8, 11]. The other is based on aromatase inhibitors and luteinizing hormone - releasing hormone agonists, which reduce the level of circulating estrogen, thereby inhibiting er activation by depriving the receptor of its ligand . Tamoxifen was already shown to inhibit proliferation and invasion of hnscc cell lines, resulting in apoptosis, which could be further enhanced upon combination with cisplatin [7678]. Thus, a therapeutic role of antiestrogens or aromatase inhibitors in the clinical management of hnscc is currently under investigation, and the results of just completed clinical trials (table 2) are eagerly awaited.however, the precise molecular roles and impact of estrogen receptor - like receptors for the onset and/or progression of head and neck cancer remain to be clarified . This knowledge will be required, in order to rationally decide whether to further investigate the potential of modern endocrine therapy also for this tumor entity . Considering the impressive benefit of endocrine therapy in breast cancer, targeting sex steroid hormone receptor as a potential therapeutic strategy is also discussed for hnscc [12, 75]. Currently, two main strategies are pursued in endocrine therapy of er - positive tumors . One is based on steroidal antiestrogens like tamoxifen, which bind to the er, block its function, and ultimately induce receptor degradation [8, 11]. The other is based on aromatase inhibitors and luteinizing hormone - releasing hormone agonists, which reduce the level of circulating estrogen, thereby inhibiting er activation by depriving the receptor of its ligand . Tamoxifen was already shown to inhibit proliferation and invasion of hnscc cell lines, resulting in apoptosis, which could be further enhanced upon combination with cisplatin [7678]. Thus, a therapeutic role of antiestrogens or aromatase inhibitors in the clinical management of hnscc is currently under investigation, and the results of just completed clinical trials (table 2) are eagerly awaited . However, the precise molecular roles and impact of estrogen receptor - like receptors for the onset and/or progression of head and neck cancer remain to be clarified . This knowledge will be required, in order to rationally decide whether to further investigate the potential of modern endocrine therapy also for this tumor entity . Nrs are associated with head and neck cancer and hence seem to be at least partially amenable for prevention and/or treatment strategies . So far, three nr groups have mainly been linked with hnscc, the retinoic acid and the peroxisome proliferator - activated and the estrogen receptors . Also, target genes activated by these nr subfamilies (table 3) have been implicated as key elements in the molecular circuits involved in head and neck cancer development and progression . Reports on other members of the nr superfamiliy are rather scarce for this tumor entity, suggesting that they have not been investigated so far . Taking the thyroid hormone receptor as an example, many studies on its relevance for various malignancies have been conducted, whereas its role in head and neck cancer, including even thyroid carcinomas, has not been analyzed in detail . Likewise, data on the cancer - related biological functions of orphan nrs are still missing for this tumor entity [7, 8]. Cancer's achilles' heel, it may be conceivable to speculate that molecules present in diet, tobacco, or beetle nut might deregulate the cell's metabolism by affecting nrs and as such contribute to head and neck carcinogenesis [27, 80]. Of note, the development of novel nr ligands with improved specificity and activity is currently intensively pursued in the area of metabolic diseases (see [7, 8, 81]). Hence, an interdisciplinary exploitation of the existing knowledge of nr pharmacobiology may result in novel hnscc treatment approaches . In sum, keeping in mind the enormous success of nr targeting therapeutics in several malignancies, a systematic investigation of nr biology as well as of its clinical relevance is highly desirable also for head and neck cancer . Together with the outcomes of current clinical trials (table 2), such improved knowledge will hopefully result in strategies with improved benefit for the patient.

Alcoholic liver disease (ald) represents a spectrum of clinical illness and morphological changes that range from fatty liver, hepatic inflammation, and necrosis (alcoholic hepatitis) to progressive fibrosis (alcoholic cirrhosis). Many of the toxic effects of ethanol in the liver have been associated with its metabolism . Ethanol oxidation generates toxic products such as acetaldehyde, and reactive oxygen species result in oxidative stress that initiates apoptosis and cell injury [25]. It is activated by endotoxin, cytokines, and oxidative stress . In unstimulated cells, nf-b is a heterodimeric complex that is sequestered in the cytoplasm by its interaction with ib family of inhibitors . When these cells are stimulated, ib is phosphorylated with subsequent release of nf-b resulting in the translocation of nf-b from the cytoplasm to the nucleus where it activates the expression of target genes [7, 8]. Activation of nf-b increased expression of proinflammatory cytokines and chemokines that were key factors in ethanol - induced liver injury rats [912]. Peroxisome proliferators activated receptors gamma (ppar) is a family of ligand - activated nuclear transcriptional factor which regulates cell differentiation, apoptosis, lipid metabolism, and inflammation . More recently, decreased expression of ppar has been found in rats with alcoholic liver fibrosis . These suggested that ppar may play an important role in the development of hepatocellular inflammation, necrosis, and fibrosis in rats with ethanol consumption . Curcumin (diferuloylmethane), an antiinflammatory and antioxidant compound, is isolated from the rhizomes of the plant curcuma longa linn . Importantly, it has been showed that curcumin suppressed the activation of nf-b in ethanol - induced liver injury in rats . Activation of ppar by curcumin resulted in inhibition of nf-b trans activating activity and increased expression of ppar at both the transcriptional and translational levels in activated hepatic stellate cells (hscs). However, it is unclear whether curcumin had any effect in early stage of ethanol - induced liver injury . Therefore, the present study determined the effect of curcumin on early stage of ethanol - induced liver inflammation and improved pathology in rats . Female sprague - dawley rats, weighing 180220 grams, purchased from the national laboratory animal center, mahidol university, salaya, nakorn pathom, were used . The rats were kept in a controlled temperature room at 25 1c under standard conditions (12-hour day - night rhythm). All rats were received well care in accordance with the ethical committee, faculty of medicine, chulalongkorn university, thailand . Curcumin in powder form (cayman chemical company, usa) is dissolved in 50% ethanol that freshly prepared for the experiment . All rats were fed with the controlled diet which contained 35% of energy from fat, 18% from protein, and 47% from carbohydrate for 4 weeks ad libitum . Group 1 (control, n = 8): rats were fed distilled water (2.0 ml) orally via an intragastric tube once per day for 4 weeks . Group 2 (ethanol, n = 8): rats were fed 50% ethanol (7.5 g / kg bw a day) orally via an intragastric tube twice a day for 4 weeks . Group 3 (ethanol + curi, n = 6): rats were fed curcumin (200 mg / kg bw) dissolved in 50% ethanol (7.5 g / kg bw a day) via intragastric tube twice a day for 4 weeks . Group 4 (ethanol + curii, n = 7): rats were fed curcumin (600 mg / kg bw) dissolved in 50% ethanol (7.5 g / kg bw a day) by using intragastric tube twice a day for 4 weeks . At the end of the study three small pieces of livers were collected, frozen in liquid nitrogen, and stored at 80c for mda analysis, sod activity, and ppar protein expression . The remaining of liver was fixed in 10% formalin solution to determine histopathology, nf-b activation, and hepatic apoptosis . After the liver samples were fixed in 10% formalin solution at room temperature, they were processed by the standard method . Briefly, tissues were embedded in paraffin, sectioned at 5 m, and stained with hematoxylin - eosin, and then picked up on glass slides for light microscopy . All fields in each section were examined for grading of steatosis and necroinflammation according to colantoni et al . Steatosis was scored as the percentage of parenchymal cells containing fat (micro- or macrosteatosis): 0 = no parenchymal cells containing fat, 1 = <20% of parenchymal cells containing fat, 2 = 2039% of parenchymal cells containing fat, 3 = 4050% of parenchymal cells containing fat, inflammation and necrosis were scored by the number of foci of inflammation and necrosis identified under low - power field of light microscope: 0 = no inflammation and necrosis, 1 = 1 focus per low - power field of inflammation and necrosis, 2 = 2 foci per low - power field of inflammation and necrosis, 3 = 3 or more foci per low - power field of inflammation and necrosis . Mda was assayed by determining the rate of production of thiobarbituric acid - reactive components . An aliquot of 0.2 ml was mixed with solution containing 20% acetic acid, 0.8% thiobarbituric acid, and 8.1% sodium dodecyl sulfate, heated in water bath at 95c for 60 minutes . The solution was centrifuged for 10 minutes at 4 000 rpm, and the absorbance of the supernatant fraction was determined at a wavelength of 546 nm . Sod was determined using the method of winterbourn, in which the light - triggered release of superoxide radicals from riboflavin leads to the formation of a blue complex through reaction with nitroblue tetrazolium . One gram of the liver was homogenized in 0.1 m phosphate buffer ph 7.4 on ice and cleared by centrifugation at 3 000 rpm at 4c for 15 minutes . The supernatant fraction was incubated in solution containing 0.067 m phosphate buffer ph 7.8, 0.1 m edta, 1.5 mm nbt and 0.12 mm riboflavin for 10 minutes in an illuminated chamber with an 18 w fluorescent lamp . Absorbance was recorded at 560 nm, and sod activity was expressed as units / mg protein . Apoptosis was measured by the identification of apoptotic nuclei in sections of liver by fragment end labeling of dna (apoptosis detection kit, chemicon, usa). In brief, the dna fragments were allowed to bind an antidigoxigenin antibody that was conjugated to a peroxidase . Diaminobenzidine (dab) was applied to develop dark brown color and then the slides were counterstained with hematoxylin . The results were expressed as the number of positive stained cells per high - power field . The liver sections were deparaffinized with xylene and ethanol for ten minutes . After water washing, sections retrieved the antigen (nf-b p65, santa cruz, usa) with citrate buffer ph 6.0 in microwave for thirteen minutes . Next, 3% h2o2 and 3% normal horse serum were performed on the slides to block endogenous peroxidase activity for five minutes and blocked nonspecific binding for twenty minutes, respectively . Then, the primary antibody used for nf-b p65, a polyclonal antibody against the p65 subunit, was applied at a dilution of 1:150 for one hour at room temperature and incubated with the secondary antibody for thirty minutes . When the development of the color with dab was detected, the slides were counterstained with hematoxylin . Under light microscopy, the positive stained cells presented dark brown in nucleus . The results were expressed as the number of positive stained cells per high - power field . Liver sample (0.1 g) was homogenized in 1 ml of lysis buffer for 30 minutes on ice and cleared by centrifugation at 12 000 rpm for 15 minutes at 4c . A 60 g of protein was applied to 10% sds - page gel, and the fractionated proteins were transferred to polyvinylidene fluoride membrane . Membrane was blocked in tbst containing 5% dry nonfat milk for 1 hour and then incubated with ppar monoclonal antibodies (1:400, santa cruz, usa) overnight at 4c . Then washed three times and incubated with secondary antibody, goat antimouse igg horseradish peroxidase (1:4,000, cayman, usa) for 1 hour . All data were presented as means and standard deviation (sd). For comparison among all groups of animals, one way analysis of variance (one - way anova) and the histologic appearance of the liver in the control group was normal (figure 1(a)). In the ethanol - treated group, the histologic features showed mild to moderate steatosis and mild necroinflammation (figure 1(b)). Rats treated with ethanol and curcumin 400 mg / kg bw a day improved the liver histopathology that showed only mild steatosis but not necroinflammation (figure 1(c)). The high dose of curcumin treatment (1,200 mg / kg bw a day) also improved the liver histopathology that showed mild steatosis and mild necroinflammation (figure 1(d)). The summary of steatosis and necroinflammation score were shown in table 1 . The level of hepatic mda, a marker of lipid peroxidation, increased significantly in ethanol - treated group as compared with control group (3.42 1.36 versus 1.44 0.24 nmol / mg protein, p <.05). Curcumin treatment (400 or 1,200 mg / kg bw a day) decreased the elevation of hepatic mda level significantly when compared with ethanol - treated group (1.43 0.14 versus 3.42 1.36 and 1.43 0.29 versus 3.42 1.36 nmol / mg protein, resp . ; our results showed that the level of hepatic sod activity of the control group was 1081.36 145.01 units / mg protein, while that of the ethanol - treated group was 1135.86 209.48 units / mg protein . In rats treated with ethanol and curcumin (400 or 1,200 mg / kg bw a day), the levels of hepatic sod activity were 966.28 139.44 and 967.84 116.66 units / mg protein, respectively . The number of apoptotic nuclei in the liver of control group was very low (0.38 0.28 cells / high - power field). In contrast, the numbers of apoptotic cells were observed frequently in centrilobular area in ethanol - treated group when compared with control group (2.43 2.68 versus 0.38 0.28 cells / high - power field, p <.05) (figures 4 and 5). There was a trend of decreased apoptosis in low dose of curcumin treatment, but the difference did not reach a statistical significance (figure 4). The number of positive stained cells in the liver of ethanol - treated group was significantly higher than control group (1.08 0.52 versus 0.04 0.04 cells / high - power field, p <.05). In contrast, curcumin treatment (400 or 1,200 mg / kg bw a day) decreased the number of positive stained cells significantly when compared with ethanol - treated group (0.15 0.02 versus 1.08 0.52 and 0.17 0.09 versus 1.08 0.52 cells / high - power field, resp . ; p <.05) (figures 6 and 7). In order to examine the change of ppar protein expression in early stage of ethanol - induced liver injury the ppar protein expression in control group was 0.57 0.19, and ethanol group was 0.68 0.16 . Rats treated with ethanol and curcumin (400 or 1,200 mg / kg bw a day) had 0.44 0.03 and 0.54 0.23, respectively . These data did not show a significant change in ppar protein expression in the liver in all groups (figure 8). Ethanol oxidation generates toxic metabolites, free radicals; and induces a state of oxidative stress which contributes to the pathogenesis of ald . Importantly, oxidation of ethanol through the cytochrome p450 2e1 (cyp 2e1) generates superoxide anion radical and hydrogen peroxide [2, 21, 22]. These free radicals are capable of damaging many cellular components such as dna, protein, and lipid . One of the characteristic features of oxidative stress is enhancement of lipid peroxidation . A number of studies have been demonstrated that ethanol intake increased the formation of lipid peroxidation product, such as mda [2426]. We found that an increase in hepatic mda level as well as pathological changes were observed in ethanol - treated group . To counteract this oxidative stress, cells have a variety of antioxidant enzymes, including sod, catalase, and glutathione peroxidase . The effects of chronic ethanol exposure on activity of sod are controversial, with reports of decrease or no changes [25, 28]. These studies may reflect variations in experimental design, diet, and duration of ethanol feeding . Decreased sod activity in ethanol fed rats was associated with enhancement of lipid peroxidation and severe pathology of liver . Therefore, the sod activity in liver did not change in early ethanol - induced liver injury . Oxidative stress can also initiate or amplify inflammation through upregulation of several genes involved in the inflammatory response . One such gene is nf-b, whose activation results in the upregulation of proinflammatory cytokines . Activation of nf-b and upregulation of cytokine production occurred in ethanol - induced liver injury and are associated with lipid peroxidation [9, 10]. Evidence was presented that curcumin prevented ethanol - induced liver injury in rats by inhibiting the expression of nf-b - dependent genes . Although, a high dose of curcumin treatment (1,200 mg / kg bw) was not better than low dose (400 mg / kg bw), the present study showed that curcumin improved ethanol - induced liver injury by reduction of oxidative stress and inhibition of nf-b activation . Ethanol - induced liver injury has been linked to oxidative stress caused by the production of reactive oxygen intermediates that cause mitochondrial dysfunction, leading to a release of proapoptotic factors such as cytochrome c that can activate caspases and initiate the apoptotic cascade in hepatocytes . Jin and coworker observed the pathological changes and investigated the correlation of hepatocyte apoptosis with cyp2e1 expression and oxygen free radical in rats with ald . Using the tunel assay, we detected a difference in apoptosis between the control and ethanol - treated group that was similar to human alcoholic hepatitis and experimental rat model of ald [3335]. Cells in centrilobular area are low o2 and nutrient supply thus the distribution of apoptotic cells is observed frequently in centrilobular area . In this study curcumin treatment did not detect a difference in hepatocyte apoptosis; however, this was a trend of decreased apoptosis in low does of curcumin treatment . This stage showed severe liver injury and hsc activation . In normal liver, hscs undergo a process known as activation, which upregulate cytokines and growth factor . For instance, platelet - derived growth factor is capable of inhibiting ppar expression via mitogen - activated protein kinase - mediated phosphorylation of ppar . Also, tnf-, inflammatory cytokine, is known to inhibit ppar expression in adipocytes and an early phase of hsc activation in liver fibrosis [38, 39], thus alcoholic liver fibrosis rats could decrease ppar expression . Our model showed only mild steatosis, necroinflammation, and no hsc activation; therefore, no change of ppar protein expression was found in ethanol - treated group . Further studies should be determined roles of ppar in different stages of ald . In conclusion, our study demonstrated that curcumin, a representative phenolic antioxidant and antiinflammmation, could improve histopathology of liver in early stage of ethanol - induced liver injury by reduction of oxidative stress and inhibition of nf-b activation . For hepatocyte apoptosis, curcumin treatment might have a trend of decreased apoptotic cells in ethanol - fed rats.

This study provides class iii evidence that more patients are seizure - free and have stopped aed treatment in the long term after resective epilepsy surgery than nonoperated epilepsy patients . In sweden, all epilepsy surgery procedures are reported to the snesur, which was initiated in 1990 . An internal control system rejects certain impossible combinations of data and regular external quality controls are performed by an independent controller . Since 2005, the follow - up has been extended from 2 years to 5, 10, and 15 years postoperatively . Snesur contains baseline information on patient's epilepsy history, preoperative seizure types and syndromes, mean monthly seizure frequency during the year preceding the presurgical investigation, aeds, preoperative investigations, psychosocial data, surgical data (type and location of surgery), histopathologic diagnoses, and postoperative complications . Two - year follow - up data cover seizure situation, aeds, and psychosocial data . The 5-, 10-, and 15-year follow - ups are structured telephone interviews regarding seizure situation, aeds, psychosocial aspects, and driving . In this study, we analyzed seizure outcome and aed medication 5 and 10 years after resective epilepsy surgery in patients who had 5- and 10-year follow - ups in 2005 to 2007 (and hence were operated on in 2000 to 2002 and 1995 to 1997). The cohort comprises the 327 patients who had resective surgery during these time periods . In 2005 to 2007, 144/176 patients operated on in 1995 to 1997 (98/116 adults and 46/60 children 18 years) had a 10-year follow - up, and 134/151 patients operated on in 2000 to 2002 (92/103 adults and 42/48 children 18 years) had a 5-year follow - up . Seventeen patients were reoperated before long - term follow - up and there were 11 deaths . Twenty - one patients (6.4%) were lost to follow - up (for details, see figure e-1 on the neurology web site at www.neurology.org). As a control group, consecutive patients who underwent presurgical investigations during the same time periods but were not operated were identified at 3 of the 6 operating centers (gteborg, uppsala, and lund). Eighty adults and 13 children out of 94 adults and 13 children underwent a cross - sectional long - term follow - up in 2008 after a mean of 9.3 years (adults) and 8.8 years (children) using the same structured telephone interview as for the surgical group . Thirteen adult patients had died (4 epilepsy - related deaths) and 1 was lost to follow - up (1%). Reasons for not having surgery were nonconclusive workup (n = 41), multifocality (n = 27), patient declined surgery (n = 12), seizure onset within eloquent cortex (n = 11), and neuropsychological reasons (n = 2). Seizure freedom (without or with aura, international league against epilepsy [ilae] class i and ii) is reported for the year preceding the follow - up except for patients with sustained seizure freedom since surgery, which is separately reported . For patients with continuing seizures or seizure relapse postoperatively, the mean monthly seizure frequency in the last year of follow - up is categorized as follows: 75% reduction in seizure frequency; 50%74% reduction in seizure frequency; 0%49% reduction in seizure frequency; and increased seizure frequency . For comparison between 2 groups, fisher exact test was used for dichotomous variables, mann - whitney u test for continuous variables, and mantel - haenszel test for ordered categorical variables . Logistic regression analysis was performed for each independent variable to predict seizure - free outcome . A forward stepwise multiple logistic regression was used to select independent predictors to outcome . Only univariate predictors attaining a p value of <0.10 this study was approved by the regional board of medical ethics at the university of gothenburg . Consent for research was obtained from all controls . For operated patients, the board considered long - term follow - up as a quality control measure not necessitating individual consent . Primary research questions were as follows: are more patients seizure - free and without aed in the long term after resective epilepsy surgery compared to nonoperated patients? This longitudinal observational study provides class iii evidence that 41% of adults and 44% of children have sustained seizure freedom in the long term after surgery compared to none of the nonoperated patients (p <0.0005). Also, 43% of seizure - free adults and 86% of seizure - free children had stopped aeds after 10 years compared to none of the nonoperated patients (p <0.0005). This study was approved by the regional board of medical ethics at the university of gothenburg . Consent for research was obtained from all controls . For operated patients, the board considered long - term follow - up as a quality control measure not necessitating individual consent . Primary research questions were as follows: are more patients seizure - free and without aed in the long term after resective epilepsy surgery compared to nonoperated patients? This longitudinal observational study provides class iii evidence that 41% of adults and 44% of children have sustained seizure freedom in the long term after surgery compared to none of the nonoperated patients (p <0.0005). Also, 43% of seizure - free adults and 86% of seizure - free children had stopped aeds after 10 years compared to none of the nonoperated patients (p <0.0005). Table 1 shows baseline characteristics of the operated patients and the controls, children (18 years) and adults . The only adult who underwent hemispherectomy at age 20 was added to the pediatric hemispherectomy group . None of the baseline characteristics differed between operated and nonoperated patients, except for number of previously tried aeds in adults (p <0.001) and iq in children (p = 0.042). Baseline data of operated patients and nonoperated controls there was no significant difference in seizure outcome between the operated patients who had follow - up after 5 years compared to 10 years . In order to enable comparison with the nonoperated group, who had a mean long - term follow - up of 9.1 years (range 514), the results from 5- and 10-year follow - ups in the operated patients were merged (figure 1). Two - year and long - term (mean follow - up time 7.6 years, range 510) seizure situation for patients after resective epilepsy surgery compared to long - term follow - up of nonoperated controls (mean follow - up time 9.2 years, range 514). Seizure - free patients include those with sustained seizure freedom with or without aura since surgery (blue) and patients seizure - free at least the last year before follow - up (green). Overall, 117 (62%) of the operated adults were seizure - free at long - term follow - up compared to 11 (14%) of the controls (p <0.001). Fifty percent (n = 93) of the operated adults had sustained seizure freedom at the 2-year and 41% (n = 78) at the long - term follow - up . None of the controls was seizure - free for the whole time period . For the children, 44 (50%) in the operated group were seizure - free the year before long - term follow - up, compared to 5 (38%) in the control group (not significant). Fifty - three percent (n = 46) of the operated children had sustained seizure freedom since surgery at the 2-year and 44% (n = 39) at the long - term follow - up, compared to none in the control group . For both adults and children, the proportion with sustained seizure freedom at long term was significantly higher compared to the nonoperated patients (p <0.0005). Details on seizure outcome for the operated patients at 2, 5, and 10 years after epilepsy surgery are shown in table e-1 . Eighty - seven percent of the adults as well as of the children who were seizure - free after 2 years were seizure - free at the 5- or 10-year follow - ups (95% confidence interval [ci] 7794 and 8095, respectively). Seizure outcomes for adults and children after different resection types are illustrated in table 2 . The most common resection type was tlr (81% in adults, 43% in children) followed by frontal lobe resection (flr) in adults (12%) and multilobe resection in children (20%). In adults, the best long - term seizure outcome was after tlr (63% seizure - free, sustained seizure freedom since surgery in 44%). In children the tlr outcome (60% seizure - free, 55% with sustained seizure freedom) was matched by the long - term hemispherectomy results (60% seizure - free, all with sustained seizure freedom). If the extratemporal procedures are summed up, 31% of adults had sustained seizure freedom at long - term follow - up, compared to 36% of the children . Seizure outcomes in the long term related to resection type the main histopathologic diagnoses were categorized as follows: hippocampal sclerosis (hs, n = 45), neurodevelopmental tumors (n = 34), low - grade astrocytomas (n = 21), cavernous hemangiomas (n = 21), malformations of cortical development (n = 52), gliosis (n = 65), and other (n = 40). Highest seizure freedom rates were seen in hs (60%, 47% sustained since surgery) and epileptogenic lesions (72%, 57% sustained since surgery) (table e-2). The univariate relationship between long - term seizure freedom and the following baseline variables was explored for both operated patients and controls: epilepsy duration in percent of life length (relative epilepsy duration, in odds ratio [or] increment units of 10%), 30 seizures / month, and presence of secondarily generalized tonic - clonic seizures (sgtcs). For the operated patients, additional variables were positive mri, resection types, and histopathology (figure 2). For the controls, the univariate associations were nonsignificant although in the same direction as for the operated patients (relative epilepsy duration, or 0.89 [95% ci 0.731.09], 30 seizures / month, or 0.88 [95% ci 0.223.47], and sgtcs, or 0.56 [95% ci 0.181.68]). In the surgical group, multivariate analysis was performed, where 30 seizures / month, or 0.40 (95% ci 0.230.69), and relative epilepsy duration, or 0.91 (95% ci 0.831.00), were negative predictors, while positive mri, or 1.96 (95% ci 1.083.55), was a positive predictor of seizure - free long - term outcome . The auc value (goodness of fit) for the final multiple model was 0.66 . For the categorical variables with more than 2 categories (resection type and histopathology), temporal lobe resections (tlr) and epileptogenic lesions (epi les) were chosen as reference categories . Ci = confidence interval; epi dur (per 10% of life) = epilepsy duration in percentage of life length, odds ratio units per 10% of life; epileptogenic lesions = gangliogliomas, dysembryoplastic neuroepithelial tumors, cavernomas, and low - grade astrocytomas; flr = frontal lobe resection; he = hemispherectomy; hs = hippocampal sclerosis; mcd = malformations of cortical development; mlr = multilobe resection; or = odds ratio; other = vascular malformations other than cavernomas, cysts, tuberous sclerosis, unknown histopathology; p / olr = parietal or occipital lobe resection; sgtcs = secondarily generalized tonic - clonic seizures . At baseline, 183 (66%) of the patients had 2 aeds, 85 (31%) had 1 aed, and 8 (3%) had no aeds . The number of aeds used by the operated seizure - free patients decreased over time (figure 3). Overall, 45 (54%) patients who were seizure - free at 10-year follow - up had stopped aeds, compared to none of the seizure - free controls (p <0.0005). More children than adults had stopped aeds (86% of the children and 43% of the adults, p = 0.002). There was no significant association between the proportion of seizure - free patients who stopped aeds and resection type . Among patients with seizures, the proportion with 2 aeds had increased from 77% preoperatively to 82% 10 years after surgery . Number of antiepileptic drugs at the start of preoperative investigations, and after 2, 5, and 10 years for patients who were seizure - free at 5- or 10-year follow - up . Table 1 shows baseline characteristics of the operated patients and the controls, children (18 years) and adults . The only adult who underwent hemispherectomy at age 20 was added to the pediatric hemispherectomy group . None of the baseline characteristics differed between operated and nonoperated patients, except for number of previously tried aeds in adults (p <0.001) and iq in children (p = 0.042). There was no significant difference in seizure outcome between the operated patients who had follow - up after 5 years compared to 10 years . In order to enable comparison with the nonoperated group, who had a mean long - term follow - up of 9.1 years (range 514), the results from 5- and 10-year follow - ups in the operated patients were merged (figure 1). Two - year and long - term (mean follow - up time 7.6 years, range 510) seizure situation for patients after resective epilepsy surgery compared to long - term follow - up of nonoperated controls (mean follow - up time 9.2 years, range 514). Seizure - free patients include those with sustained seizure freedom with or without aura since surgery (blue) and patients seizure - free at least the last year before follow - up (green). Overall, 117 (62%) of the operated adults were seizure - free at long - term follow - up compared to 11 (14%) of the controls (p <0.001). Fifty percent (n = 93) of the operated adults had sustained seizure freedom at the 2-year and 41% (n = 78) at the long - term follow - up . None of the controls was seizure - free for the whole time period . For the children, 44 (50%) in the operated group were seizure - free the year before long - term follow - up, compared to 5 (38%) in the control group (not significant). Fifty - three percent (n = 46) of the operated children had sustained seizure freedom since surgery at the 2-year and 44% (n = 39) at the long - term follow - up, compared to none in the control group . For both adults and children, the proportion with sustained seizure freedom at long term was significantly higher compared to the nonoperated patients (p <0.0005). Details on seizure outcome for the operated patients at 2, 5, and 10 years after epilepsy surgery are shown in table e-1 . Eighty - seven percent of the adults as well as of the children who were seizure - free after 2 years were seizure - free at the 5- or 10-year follow - ups (95% confidence interval [ci] 7794 and 8095, respectively). Seizure outcomes for adults and children after different resection types are illustrated in table 2 . The most common resection type was tlr (81% in adults, 43% in children) followed by frontal lobe resection (flr) in adults (12%) and multilobe resection in children (20%). In adults, the best long - term seizure outcome was after tlr (63% seizure - free, sustained seizure freedom since surgery in 44%). In children the tlr outcome (60% seizure - free, 55% with sustained seizure freedom) was matched by the long - term hemispherectomy results (60% seizure - free, all with sustained seizure freedom). If the extratemporal procedures are summed up, 31% of adults had sustained seizure freedom at long - term follow - up, compared to 36% of the children . Seizure outcomes in the long term related to resection type the main histopathologic diagnoses were categorized as follows: hippocampal sclerosis (hs, n = 45), neurodevelopmental tumors (n = 34), low - grade astrocytomas (n = 21), cavernous hemangiomas (n = 21), malformations of cortical development (n = 52), gliosis (n = 65), and other (n = 40). Highest seizure freedom rates were seen in hs (60%, 47% sustained since surgery) and epileptogenic lesions (72%, 57% sustained since surgery) (table e-2). The univariate relationship between long - term seizure freedom and the following baseline variables was explored for both operated patients and controls: epilepsy duration in percent of life length (relative epilepsy duration, in odds ratio [or] increment units of 10%), 30 seizures / month, and presence of secondarily generalized tonic - clonic seizures (sgtcs). For the operated patients, additional variables were positive mri, resection types, and histopathology (figure 2). For the controls, the univariate associations were nonsignificant although in the same direction as for the operated patients (relative epilepsy duration, or 0.89 [95% ci 0.731.09], 30 seizures / month, or 0.88 [95% ci 0.223.47], and sgtcs, or 0.56 [95% ci 0.181.68]). In the surgical group, multivariate analysis was performed, where 30 seizures / month, or 0.40 (95% ci 0.230.69), and relative epilepsy duration, or 0.91 (95% ci 0.831.00), were negative predictors, while positive mri, or 1.96 (95% ci 1.083.55), was a positive predictor of seizure - free long - term outcome . The auc value (goodness of fit) for the final multiple model was 0.66 . For the categorical variables with more than 2 categories (resection type and histopathology), temporal lobe resections (tlr) and epileptogenic lesions (epi les) were chosen as reference categories . Ci = confidence interval; epi dur (per 10% of life) = epilepsy duration in percentage of life length, odds ratio units per 10% of life; epileptogenic lesions = gangliogliomas, dysembryoplastic neuroepithelial tumors, cavernomas, and low - grade astrocytomas; flr = frontal lobe resection; he = hemispherectomy; hs = hippocampal sclerosis; mcd = malformations of cortical development; mlr = multilobe resection; or = odds ratio; other = vascular malformations other than cavernomas, cysts, tuberous sclerosis, unknown histopathology; p / olr = parietal or occipital lobe resection; sgtcs = secondarily generalized tonic - clonic seizures . At baseline, 183 (66%) of the patients had 2 aeds, 85 (31%) had 1 aed, and 8 (3%) had no aeds . The number of aeds used by the operated seizure - free patients decreased over time (figure 3). Overall, 45 (54%) patients who were seizure - free at 10-year follow - up had stopped aeds, compared to none of the seizure - free controls (p <0.0005). More children than adults had stopped aeds (86% of the children and 43% of the adults, p = 0.002). There was no significant association between the proportion of seizure - free patients who stopped aeds and resection type . Among patients with seizures, the proportion with 2 aeds had increased from 77% preoperatively to 82% 10 years after surgery . Number of antiepileptic drugs at the start of preoperative investigations, and after 2, 5, and 10 years for patients who were seizure - free at 5- or 10-year follow - up . Since long - term results of epilepsy surgery cannot be obtained from rcts, observational studies with defined quality criteria are needed . A number of requirements for studies on the prognosis after epilepsy surgery have been suggested . Studies based on snesur meet many of these requirements, with a prospective design, representative study populations, a standard protocol for outcome measures, and prolonged longitudinal follow - up with few patients lost to follow - up . We found 63% of adults and 60% of children to be seizure - free after tlr at the long - term follow - up and most had continuous seizure freedom since surgery (adults 41%, children 44%). As expected, the results were not as good after flr, with 44% of adults seizure - free (sustained in 35%), and in children 27% seizure - free (all sustained). This is still substantially better than for the patients who could not be operated (14% seizure - free adults, none sustained and 38% seizure - free children, none sustained). The adults who underwent other types of resections (posterior or multilobar) were few, and only 3 had sustained long - term seizure freedom . Children who had undergone posterior resections or hemispherectomies had long - term seizure freedom in 55% and 60%, respectively (sustained in 46% vs 60%). Long - term outcome after resective epilepsy surgery is often reported cross - sectionally . In a meta - analysis from 2005 based on 78 studies, 66% of tlr patients, 46% of parietal or occipital lobe resection patients, and 27% of flr patients were seizure - free at follow - up 5 years postsurgery, but the authors point out that few studies reported sustained seizure freedom from surgery . A number of recent longitudinal long - term outcome studies report sustained seizure freedom after tlr . Sustained seizure freedom is reported as engel 1, engel 1a, or engel 1a+b and in a few studies as ilae class i and ii . Sustained seizure freedom around 5 years postoperatively varies between 46% and 55% and 60% to 80% . Among the studies with moderate rates of sustained seizure freedom, 2 of 4 are prospective, and our prospective study with 41% sustained seizure freedom at long - term follow - up after tlr in adults and 44% in children supports these results . A number of studies report cross - sectional long - term follow - up of pediatric epilepsy surgery, with seizure freedom in 50% to 82% of patients 5 to 12 years after tlr . Only one study is longitudinal and reports sustained seizure freedom (engel 1) in 54% of patients 5 years after tlr . There are few retrospective reports of long - term outcomes in patients after flr or other extratemporal resections . Sustained seizure freedom 5 years after surgery varies in these studies from 15% and 27% to 52%, which can be compared to 31% (adults) vs 36% (children) in our cohort of 86 patients with varying extratemporal resections . Long - term reports from population - based cohorts are sparse . In a retrospective cross - sectional comprehensive audit from ireland, national long - term seizure outcome data were reported . Forty - four percent of patients were seizure - free 10 years after resective surgery, but the total number of operated patients could not be accounted for . A recent norwegian retrospective questionnaire study in children with a response rate of 70% reported 58% seizure - free after a mean of 7 years . However, sustained seizure freedom was not reported in these studies . In a recent systematic review, 20 studies comparing surgical results to those of a nonsurgical control group were identified; 13 had a follow - up of at least 4 years . The controls were in general patients evaluated for surgery who for various reasons were not operated . Overall 44% of surgical patients were seizure - free at follow - up (in 90% of the studies, seizure freedom was reported at last follow - up or last year) compared to 12% of medically treated patients . This is well in line with our findings of 58% seizure - free at last year of follow - up in the surgical group and 17% in the nonsurgical group, children and adults together . It must be remembered that patients who after preoperative investigations are considered ineligible for epilepsy surgery are not comparable to surgically treated patients, and they may have another disease course . It is difficult though to identify other reasonable controls for long - term follow - up studies, and from a clinical point of view they match the operated patients in many baseline variables . Predictors for seizure freedom in the long term (4 years) have been sought by several investigators . While some found no remaining predictors in multivariate analysis, the most commonly identified predictors are sgtcs at baseline and epilepsy duration or age at surgery . We found high baseline seizure frequency and relative epilepsy duration to be negatively and positive mri to be positively related with long - term seizure freedom . Epilepsy duration before undertaking presurgical investigation has repeatedly been shown not to have shortened significantly over the years . These results from long - term outcome studies underline the importance of earlier identification of good candidates for resective epilepsy surgery . There is no common swedish protocol for discontinuation of aeds in seizure - free patients . Most adults continue aeds the first 2 postoperative years; further decisions about withdrawal are taken individually . We found that 43% of seizure - free adults and as many as 86% of seizure - free children had discontinued aeds after 10 years . The reasons for this difference are unknown; possibly, social consequences of seizure relapse are of greater importance to adults (e.g., driving or occupation), who therefore may choose to continue aeds . By contrast, more concern about adverse effects of aeds on the developing brain in children may lead to earlier decisions about withdrawal . The proportion of seizure - free adults and children in whom aeds have been withdrawn after successful epilepsy surgery varies widely across studies . In a meta - analysis from 2007, 9 studies were identified and a pooled analysis showed that 27% of seizure - free children and 19% of seizure - free adults had discontinued aeds at a mean follow - up of 7 years . However, in an indian study, aed withdrawal was systematically planned for all seizure - free patients after tlr and was successful in 63% of 258 patients who were followed for 5 years . Very similar to our results in children, a canadian study found that 82% of seizure - free children had stopped aeds 10 years after surgery . Possible reasons for these differences in patient decisions about aed cessation may include socioeconomic and cultural differences, as well as how doctors inform their patients, which partly depends on their own assessment of recurrence risk . The timetostop study showed that early withdrawal of aed in children did not influence the long - term seizure outcome but could unmask incomplete surgical success sooner, and the authors suggest that it is now time to plan an rct of aed withdrawal after epilepsy surgery in children . Like most, if not all, observational outcome studies after epilepsy surgery, we had no masking in seizure outcome assessment . Our nonoperated control group was recruited from the 3 largest of the swedish epilepsy surgery centers and is hence not a national sample . On the other hand, we have a regional referral system and it is reasonable to assume that this sample is representative of the population of patients who are evaluated for surgery in sweden but not operated . Patients need detailed counseling before deciding to undergo irrevocable brain surgery . Realistic expectations concerning long - term outcomes are part of the information they need to consider . This prospective population - based study contributes to this knowledge and supports seizure outcome data from prospective single - center studies, and also shows that a greater proportion of seizure - free patients than in most studies had stopped aed treatment 10 years after surgery . Funded by grants from the swedish research council (grant 521 - 2011 - 169), the sahlgrenska academy at gothenburg university through the lua / alf agreement (grant alfgbg137431), the gothenburg foundation for neurological research, the gothenburg medical society, the margaretahem foundation, and an unconditional research grant from glaxosmithkline to anna edelvik . K. malmgren served on an educational advisory board for ucb and has received speaker's honoraria from ucb.

The prevalence of obesity among adolescents in the united states has increased dramatically in the past 30 years, with particularly high rates among hispanic, african american, and native american youth [14]. Currently 33.6% of all adolescents 1219 years of age in the united states are either overweight or obese . Although the causes of obesity are complex, it is widely recognized that poor nutrition and physical inactivity play important roles . For this reason, public health interventions targeting youths frequently focus on health promotion programs in schools [69], as well as calling for nutrition and exercise counseling in the health care setting [1012]. The potential for physicians to influence behavioral changes among patients through simple nutrition and exercise advice, as opposed to more time - intensive counseling is crucial . Some studies have documented the value of physician - counseling either used as a stand - alone strategy or as part of a coordinated effort to help patients make changes in their diet and physical activity patterns . National health organizations have also recognized the importance of clinician counseling and have called for an increase in the provision of both nutrition and exercise counseling given to adolescents during physician visits [15, 16]. The surgeon general's recent vision for a healthy and fit nation (2010) encourages clinicians to recommend healthy eating and increased physical activity to their patients and recommends training for clinicians and health care students on effective ways to counsel patients on lifestyle behavior change . In california, where adolescent overweight and obesity rates are comparable to national levels, major programmatic and policy responses aimed at obesity prevention in adolescents have been undertaken at schools to limit nonnutritious foods and beverages sold on campus and in communities to increase access to nutritious foods and safer places to exercise [8, 9, 19]. However, little is known about the frequency of weight - related counseling given to different racial / ethnic groups or those with limited access to care, over the course of treating or preventing obesity in the health care environment . A recent study found that race was an important factor that explained the prevalence of nutrition or physical activity counseling among california adolescents: african americans compared to whites were more likely to receive nutrition counseling, while hispanics compared to whites were more likely to receive both nutrition and physical activity counseling . With regards to insurance type, data suggests that california adolescents who are uninsured or who qualify for low - cost / free insurance are at greatest risk for overweight or obesity, yet a recent national study found that adolescents with private insurance generally receive more counseling, compared to those who have low - cost insurance . Changes in the frequency of obesity - related counseling overtime by race / ethnicity or insurance type are yet to be examined . Previous research in california suggests that physician obesity - related counseling has been declining, with overall counseling rates between 2003 and 2007, shifting from 75% to 59% for nutrition and 74% to 60% for physical activity . Insufficient time, lack of resources, inadequate reimbursement, and patient noncompliance are typically cited as barriers to provision of routine advice by health care practitioners . The objectives of this current study were to document trends from 2003 to 2009 in either nutrition or exercise counseling or a combination of both among california adolescents by race / ethnicity and by insurance type . These findings can provide guidance for policies and programs in a state with high rates of adolescent overweight or obesity and large ethnic populations at particular risk . Further, we hypothesized that physicians would favor dietary counseling over exercise counseling when providing counseling . In an earlier study, stafford et al . Found that physicians offered dietary counseling to obese patients 41.5% of the time, while exercise counseling was offered only 32.8% of the time . Among healthy weight participants, branner and colleagues found higher rates of nutrition compared with exercise counseling among children and adolescents (42.1% and 26.1%, resp . ). Data demonstrating trends in nutrition and exercise counseling by race / ethnicity and by insurance type were obtained using four biennial california health interview surveys (20032009), the largest state surveys in the united states . The california health interview survey (chis) is a two stage sampling, weighted, random digital dialing telephone survey, representative of the california noninstitutionalized population . Within households, an adult and adolescent were randomly selected and interviewed by trained chis interviewers; adolescents were directly interviewed . The chis program obtained informed consent from all individuals participating in the survey and this current study was deemed exempt or waived for human subjects review by the university of california, berkeley, institutional review board . In this study, obesity - related counseling refers to simple advice about nutrition and/or exercise practices, as opposed to more time - intensive counseling . Adolescents self - reported whether they discussed nutrition or exercise habits with their physician at their last routine exam: when you had your last routine physical exam, did you and a doctor talk about nutrition or healthy eating? And when you had your last routine physical exam, did you and a doctor talk about exercise or physical activity? Data were analyzed using stata version 10, with the svy module to account for weighting and the raking method in variance estimation . Obesity - related counseling proportions are presented graphically by race / ethnicity and by insurance type for the period 20032009 . To better represent the obesity - related counseling construct, we categorized this variable as respondents having no discussions of nutrition or exercise with their physician, discussing either nutrition or exercise, or discussing both nutrition and exercise with their physician . Participants' self - reported their weight and height, which were used to generate the centers for disease control and prevention (cdc) bmi age and gender specific percentiles, categorized into underweight (<5th percentile), normal weight (5th<85th percentile), overweight (85th<95th percentile), and obese (95th percentile). In addition, insurance type variables (uninsured, medicaid, healthy families, employer - based, privately owned insurance, and other public insurance) were collapsed into the categories (uninsured, low - cost / free, employer - based, and private insurance). Medicaid is the united states health insurance program for certain low - income individuals and families, which is jointly funded by state and federal governments, while healthy families is a low - cost health insurance program for children and adolescents who do not have health insurance and who do not qualify for medicaid . Table 1 presents the study sample characteristics, using the chis (2009). Participants ranged in age from 12 to 17 years, with 51.0% being male and 49.0% being female . The sample consisted primarily of hispanics (49.3%) and non - hispanic whites (33.5%). Most adolescents had some form of health insurance, with almost 60% being covered through their parents'/guardians' employer - sponsored health insurance . Less than half of all adolescents (44.8%) were at or above 300% of the federal poverty level . Based on self - reported data, 28.7% of california adolescents were either overweight or obese, while 48.2% of all california adolescents received counseling on both nutrition and exercise subjects (table 1). The majority of respondents (84.7%) reported having a physical exam within the past year (table 1). Previously published data indicate how california adolescent demographics have changed from 2003 to 2007 . Figure 1 and table 2 present data for obesity - related counseling stratified by race / ethnicity . When examining nutrition or exercise counseling separately for the period from 2003 to 2009, african americans generally reported higher levels of nutrition than exercise counseling, while whites generally reported higher levels of exercise than nutrition counseling . Hispanics generally reported higher levels of nutrition than exercise counseling during 20032005, after which counseling levels remained consistent . Overall, trends show that counseling declined between 2003 and 2009 for all groups, except for hispanics and whites which started to increase again after 2007; american indians / alaska natives reported a sharp decline in 2009 . Between 2003 and 2009, the proportion of adolescents who reported counseling on both nutrition and exercise decreased from 66.8% to 53.7% among hispanics; from 60.7% to 15.1% among american indians / alaska natives; from 61.7% to 33.4% among asians; from 58.8% to 42.9% among african americans; and from 60.0% to 46.2% among whites (figure 1). Figure 2 and table 3 present data for obesity - related counseling stratified by insurance type . Those who had private insurance generally received exercise counseling more frequently than nutrition counseling over the study time period . There appeared to be no imbalance in frequency of nutrition or exercise counseling for the uninsured, low - cost / free or employer - based groups, except in 2003 when adolescents who were underinsured or had low - cost insurance reported more nutrition than physical activity counseling . Counseling appeared to decline from 20032009 among all insurance types, although after 2007, a slight increase was observed for the low - cost / free insurance group . Among those who were uninsured counseling declined from 70.8% to 42.2%; among the low - cost / free group, counseling declined from 64% to 53.4%; among the employer group, counseling declined from 61.8% to 46.6% and among the private insurance group, counseling declined from 58.9% to 39.8% (figure 2). As early as the 1950s, the american medical association council on food and nutrition cited the benefits of nutrition counseling, as well as inadequacies in nutrition education in u.s . Medical schools . Further, counseling has been shown to be valuable in helping patients to change their behavior and to achieve weight loss and can be even more beneficial if used as part of a coordinated approach with health education materials . Kreuter and colleagues reported that patients who received a combination of health education materials, followed by physician counseling were 51% more likely to increase their leisure time physical activity, and 35% more likely to reduce fat from dairy sources at follow - up . Some groups that need obesity prevention counseling the most may still be missing out, including american indians / alaska natives, african americans, and the uninsured . Time trend findings from 2003 to 2009 indicate that nutrition and exercise counseling decreased for all racial / ethnic groups except for hispanics and whites, for whom it started to increase after 2007 . Our findings also suggest that counseling levels in california for racial / ethnic groups and for patients with different types of insurance are generally higher compared to national levels . The higher counseling levels reported in this study, compared with national figures, may have been due to fewer barriers or more public health awareness of the obesity epidemic in california and stronger health care leadership . Previously reported barriers to counseling include insufficient reimbursement rates, lack of time, lack of training for medical providers in policy advocacy related to improved nutrition and activity environments, and the need for information on evidence - based obesity - related messages and referral networks for nutrition counseling . When examining nutrition or exercise counseling separately during the study period (20032009), interesting differences were found by race / ethnicity: hispanic and african american chis adolescents generally reported higher levels of nutrition than exercise counseling, while whites generally reported higher levels of exercise, than nutrition counseling . Meanwhile, participants who had private insurance generally received more exercise than nutrition counseling during the study period . Further research is needed to investigate the underlying factors that may explain the differences in the findings for racial / ethnic groups . The chis surveys were only able to identify the existence of discussions or conversations that adolescents may have had with their physicians regarding nutrition and exercise messages, but were not able to ascertain the depth of these discussions . Given the limited time physicians have in working with patients, it is unlikely that any advice given would be in - depth psychological advice . Further, it is difficult to ascertain whether these conversations were initiated by the physician or the patient . These data were unable to measure specific evidence - based obesity - related messages (limiting sugar sweetened beverages, increasing fruit and vegetables, reducing television viewing, and increasing moderate - to - vigorous physical activity) [7, 29]. The potential for recall bias also exists since adolescents were asked to self - report nutrition and/or exercise data that occurred during their last physical exam, however, most adolescents (84.7%) had a physical exam at their physician visit within the past year . This is the first study to examine trends in obesity - related counseling by race / ethnicity and by insurance type among california adolescents . Additionally, this is one of the first studies to examine trends in counseling among american indians / alaska natives, a group that is also disproportionately affected by overweight and obesity [3, 4]. The findings from our study have demonstrated that the downward trend in obesity prevention counseling in california among racial / ethnic groups and health insurance groups has changed course and has begun to increase . Future analysis of the biennial chis surveys will indicate if the trend continues in this direction . It is widely accepted that obesity prevention should follow a socioecological approach, combining multiple interventions tailored to specific demographic groups . While a vast amount of work has already been conducted in california to implement population - wide obesity prevention policies in schools and in low - income communities, this momentum must also be applied to california primary health care settings . However, for physician - based counseling to continue to increase among the general adolescent population and among vulnerable high - risk groups in particular, this will require institutional and policy changes . Research on effective ways to support clinicians' use of the aforementioned evidence - based obesity - related messages is still in its infancy, although preliminary data from clinic - based obesity interventions have begun to show promising results . At least one us state (washington) has adopted clinic - based programs to address the childhood obesity epidemic by establishing partnerships with hospitals, health care plans, and community - based organizations . Other states may consider adopting such a program in the primary care setting in order to build on obesity prevention programs and policies previously implemented in schools and low - income communities.

Student perception of their dental school experience is an essential measure of the success of dental education . Undergraduates' feedback and suggestions are very important for improving the curriculum and learning process . This information also helps determine the students' preferences regarding different elements of their educational experience . Endodontics teaching can be considered complex, difficult, and stressful because of the complex anatomy of the root canal system, responsibility toward patients, and low self - confidence . However, teaching endodontics in recent years has improved as a result of the development of knowledge, techniques, and materials . Dental students at taibah university, saudi arabia, take a preclinical full - year endodontic course in the 3 year of their 6-year bachelor of dental surgery degree . The course consists of 28 h of theoretical lectures and 28 3-h laboratory sessions, during which they perform technical aspects of root canal treatment on extracted single- and multi - rooted teeth . There is a one - semester clinical endodontic course in the 4 year that consists of 14 theoretical lectures and fourteen 3-h clinical sessions during which students treat single- and multi - rooted teeth . In the 5 year, endodontic treatments are performed as part of a comprehensive dentistry care course under the supervision of specialists . This study examined the endodontic experiences, perceptions of endodontic practice, and self - rated confidence of dental students enrolled in taibah university, saudi arabia . Study approval was obtained from the research ethics committee of the college of dentistry, taibah university (reference number: tucdrec/20160107/alrahabi). This study enrolled 41 undergraduate dental students registered in endodontic courses in the 2015 academic year at the college of dentistry, taibah university, saudi arabia . Participation was voluntary, and students were informed that they could refuse participation . A questionnaire was distributed to 19 4-year and 25 5-year students in the final month of the academic year after they had had the maximum amount of training . The questions evaluated self - confidence at performing nonsurgical root canal treatment and experiences in this discipline . The level of confidence was classified using a 5-point scale as very confident, confident, neutral, not very confident, or not at all confident . To compare results, the chi - square test and mann whitney u - test were used . Statistical analyses were carried out using spss version 20.0 (spss, chicago, il, usa). Statistical significance was set at p <0.05 . The overall response rate was 93%, with 18 of the 19 4-year students and 23 of the 25 final - year students returning questionnaires . Table 1 shows the results for the first three questions (first endodontic case treated, level of case difficulty, and number of endodontic treatments performed by the student). First endodontic case treated, level of case difficulty, and number of endodontic treatments performed self - confidence of 4th- and 5th - year dental students regarding the steps of nonsurgical root canal treatment levels of confidence in the different steps of root canal treatment confidence levels differed significantly between 4- and 5-year students in the following steps of root canal treatment: determining the working length, taking and interpreting radiographs during root canal treatment, evaluating the quality of root canal obturation, and recalling the patients periodically in the correct manner . Table 3 summarizes the results of the last question regarding suggestions to improve the teaching of endodontic courses . In dentistry, evaluating competence is an important step toward validating the quality of graduating dentists, although the relationship between the self - confidence and clinical competence of medical students is not fully understood . Increasing the confidence of medical students should increase their competence in clinical practice . One way to standardize education is to poll students using questionnaires to help in the assessment, evaluation, and improvement of education . This study obtained information about the confidence of undergraduate dental students at taibah university, saudi arabia regarding endodontics . It revealed that an upper incisor 78% was the most common tooth first treated by students . Upper incisor root canal treatment is relatively easy, and this should encourage students . In another study, most of our students described the experience with the first case as okay 78%, while 7.4% described it as easy and 14.6% described the first case as difficult, possibly because the first case for those students involved a molar or premolar . In other studies, students considered molars to be the most difficult tooth to treat . In our study, the maximum number of teeth treated in the 4 year was four cases, by 66.7% of the students, while the maximum number of teeth treated in 5 year was 11 cases, by 8.8% of the students . The number of teeth treated by students in the 4 and 5 year did not meet the recommendations of the european society of endodontology 2001 guidelines, which advised that for adequate competency a student should complete root canal treatments in 20 teeth . Although the european society of endodontology published new undergraduate curriculum guidelines for endodontology in 2013, these focused on the quality and consistency of student performance more than simply the quantity of clinical exposure . However, one study reported that 81% of the students in 48 dental schools in the european union achieved the minimum number of root canal treatments required for their graduation: the number of treated cases ranged between 3 and 80 canals, and the average was 17 canals . In our study, we observed that confidence varied according to both the year the student was in and the practical steps of nonsurgical root canal treatment . Both groups reported relatively good confidence, although there were significant differences between 4 and 5 year students regarding some steps . Fourth - year students were more confident than 5-year students in the following: determining the working length, dealing with x - rays during root canal treatment, evaluating root canal obturation, and recalling patient at the correct time . This might be because there were fewer 4-year students in the sample, there are fewer requirements in 4 year, and strict supervision by supervisors helped 4-year students more than 5-year students . Confidence regarding working length determination was low in both 4- and 5-year students . This might be the result of the root canal anatomy, which many dental students find difficult to learn because of its variation among individuals . The reduction in confidence regarding endodontic radiology, the evaluation of root canal obturation, and determining the correct recall period probably results from insufficient clinical exposure . Murray et al . Wrote that a lack of clinical exposure in the undergraduate curriculum reduces the confidence that develops with clinical practice . Students' suggestions for improving the teaching of endodontics focused on two major issues: using rotary nickel - titanium (niti) files during treatment and increasing credit hours for the endodontic course . Introducing advances in endodontics into undergraduate training, such as niti rotary instruments, may improve the clinical experience of students and their self - confidence because it will help increase the numbers of cases treated . Believe that introducing niti rotary instruments into the undergraduate dental curriculum would be safe and improve endodontics teaching because inexperienced operators can learn to use rotary instruments adequately with brief training . Nevertheless, another study found that intensive preclinical training is a prerequisite for using niti rotary instruments . Changing the methods of teaching endodontics so that students can complete root canal treatment more easily and quickly, with minimal procedural accidents, will improve clinical outcomes . Low self - confidence can be ameliorated by increasing clinical exposure, which will help students to obtain the necessary skills through experience . Fourth- and fifth - year dental students at taibah university, saudi arabia, are confident regarding root canal treatment, although they report lower confidence in some steps of the root canal treatment process . Endodontics education should be improved by increasing preclinical and clinical sessions and using new teaching methods that introduce recent advances in endodontics in the undergraduate curriculum.

A 36-year - old woman complained of an insidious onset of generalized myoclonus that first became apparent at age 27 years . She had no perinatal problems and her development was normal in childhood and juvenile periods . Adult - onset myoclonus had worsened progressively from right hand to four extremities, tremulous voice and gait disturbance developed after 3 years from disease onset, and she could not continue working as a nurse . She had no history of febrile convulsions or seizure, infectious disease in the central nervous system, exposure to toxic materials, or intake of herbal drugs . Her younger brother aged 40 years old also had progressive generalized myoclonus, which was detected 6 years ago at age 34 years (figure 1). The patient was alert and oriented, and her mini - mental state examination score was 30 . She did not have gaze palsy, and her vision and hearing were normal; however, her voice was tremulous and generalized positive myoclonus was observed at four extremities and body . Negative myoclonus, dystonia, tremor, and rigidity were not detected, and both motor and sensory functions were intact . There was no evidence of cerebellar dysfunction, and she did not have an ataxic or parkinsonian gait, although she staggered slightly because of myoclonus . Examination of the eyes revealed cherry - red spots (figure 2), but her electroencephalogram was normal . No white matter lesion or cerebellar atrophy was detected in an mri of her brain (figure 3). Neuraminidase, hexosaminidase a, and -galactosidase activities in the leukocytes and cultured fibroblasts in patient and younger brother were normal . The cherry - red spot is a pale perifoveal ring that develops when large deposits of lipid, sphingolipid, or oligosaccharide material accumulate in the ganglionic cells at the macula.2 this is a characteristic finding in storage diseases, including the sialidoses, gm1 and gm2 gangliosidoses, neuronal ceroid lipofuscinosis, niemann - pick disease (groups a through d), farber s lipogranulomatosis, and metachromatic leukodystrophy . Interestingly, niemann - pick disease, farber s lipogranulomatosis, and metachromatic leukodystrophy are not associated with myoclonus . Moreover, the patient in this report did not have typical findings of these 3 diseases such as the organomegaly, cognitive impairment, and gaze palsy seen in niemann - pick disease4; the hoarseness, arthritis, and subcutaneous nodules seen in farber s lipogranulomatosis; or the abnormal brain mri findings in metachromatic leukodystrophy . In ceroid lipofuscinosis, sialidosis, gm1 and gm2 gangliosidoses, myoclonus, and maculopathy (e.g. A cherry - red spot) may coexist . Although they are quite similar in appearance, the macular abnormality seen in patients with neuronal ceroid lipofuscinosis (which has been described as bulls - eye maculopathy) can be distinguished from the cherry - red spot by color and shape, as well as by the decreased visual acuity and visual - field restriction that are common in neuronal ceroid lipofuscinosis.5 moreover, adult - onset lipofuscinosis has an autosomal dominant inheritance, rather than the autosomal recessive pattern seen in this patient.6 gm1 gangliosidosis results from a deficiency of -galatosidase; the adult form (type 3) presents as a slowly progressive dementia with prominent parkinsonian features and extra - pyramidal dysfunction, particularly dystonia.7 gm2 gangliosidosis results from a deficiency of hexosaminidase a; the late form (with an onset during adolescence and young adulthood) may be characterized by cognitive dysfunction, cerebellar dysfunction, upper and lower motor neuron involvement, and extrapyramidal dysfunction.8 because -galatosidase and hexosaminidase activity was normal in this patient, because she has a normal level of intelligence, and because no other prominent pyramidal or extrapyramidal dysfunction was detected, we might not diagnose this patient as gm1 and gm2 gangliosidoses . Sialidosis is an inherited, autosomal recessive disease associated with a neuraminidase deficiency.9 it has 2 major clinical manifestations: type i (late, adult onset) and type ii (early, infantile onset). Type i sialidosis is typically found in patients aged 8 to 25 years and is characterized by cherry - red spot myoclonus, seizure, neuropathy, corneal clouding, and difficulty walking, but with normal vision and intelligence (obrien, 1978). Type ii sialidosis is characterized by dysmorphism, myoclonus, mental retardation, ocular cherry - red spots, and hepatosplenomegaly . The patient in this report may present clinical evidence of type i sialidosis, but her laboratory findings do not support this diagnosis; activities of the neuraminidase were normal . A similar case of progressive myoclonic epilepsy has been reported.10 differences from the previous report which described a patient with progressive myoclonic epilepsy, cherry - red spots and negative enzyme deficiency were theses; 1) the onset - age was older than the previous report (27 versus 13 years, adult versus juvenile - onset); 2) the patient had a sibling with same disease, which indicated she had inherited disease, but the patient in previous report did not have familial history; 3) the patient in our report did not have a history of seizure, and it is different from the patient with myoclonic epilepsy . Although the cause of cherry - red spot myoclonus is not clear, to our knowledge, this is the first report of adult - onset familial cherry - red spot myoclonus caused by an unknown type of lysosomal storage disease in korea.

With the worldwide spread of mycobacterium tuberculosis (mtb) strains resistant to both isoniazid and rifampicin (rif), multidrug - resistant (mdr) tuberculosis (tb) has become a major public health problem posing formidable challenges due to its complex diagnostic and treatment requirements . This highlights the need for having rapid molecular diagnostic techniques which could help facilitate early diagnosis and appropriate delivery of anti - tubercular therapy . Xpert mtb / rif assay (cepheid), a real - time automated nucleic acid amplification system is one such technique which detects mtb as well as mutations that confer resistance to rif in> 2 h. it was installed in our institute (guru gobind singh medical college and hospital, faridkot) in october 2013 under revised national tuberculosis control programme (rntcp) tb xpert project supported by who - stop tb partnership - unitaid . Rif is a principle first line anti - tb drug and rif resistance is a surrogate marker for mdr - tb . The genetic basis of rif resistance (in approximately 95% cases) is the presence of mutations in 81 bp rif resistance determining region (rrdr) of the rpob gene, corresponding to codons 507533 (escherichia coli numbering system), which codes for a beta subunit of rna polymerase of mtb . Studies conducted in diverse geographical areas have shown that the burden of mdr - tb and the mutations responsible for drug resistance vary from country to country and region to region . However, there is not enough of information about this important aspect from north india and the data from malwa region of punjab is almost lacking . Therefore, this retrospective study was conducted to determine the frequency of rif resistance and rpob gene mutations among the suspected tb / mdr cases of malwa region of punjab (districts: faridkot, fazilka, firozpur, moga, bathinda, and muktsar - area approximately 14,981 km), using xpert mtb / rif assay . Knowledge of the pattern of mutations present in rif - resistant isolates could provide insight into the epidemiology of rif - resistant mtb isolates of this particular area . A total of 1612 sputum specimens which originated from patients with suspected mdr pulmonary tb of malwa region of punjab were received in microbiology laboratory, under rntcp for xpert mtb / rif assay between october 2013 and february 2015 . The samples were subjected to ziehl - neelsen (zn) staining and xpert mtb / rif assay . The xpert mtb / rif assay was performed directly on sputum specimens using the xpert mtb / rif assay g4 version 5 and the software version 4.4a as per the manufacturer's instruction (cepheid, sunnyvale, ca, usa). Briefly, it consisted of inactivation of the sputum specimen with naoh and isopropanol (sample reagent) used in 1:2 ratio for 15 min . The mixture was then introduced into the xpert mtb / rif cartridge and loaded into the xpert mtb / rif instrument for dna extraction and amplification of 192 bp segments of the rpob gene . The detection consists of hybridization of the amplicon with five overlapping probes complementary to the rpob core the reports were communicated electronically to the concerned district tb officer and drug - resistant tb center . Chi - square test of proportions was used to identify a significant difference between two or more groups, and p <0.05 was considered as statistically significant . To determine the association of various epidemiological characteristics of the patients to rif - resistant tb, odds ratio (or) and 95% confidence interval (ci) were calculated using spss statistical software version 20.0.0 (spss inc ., the relationship of smear positivity grade with the relative bacterial burden and xpert mtb / rif ct value (given in genexpert operator's manual by cepheid) was also analyzed . Chi - square test of proportions was used to identify a significant difference between two or more groups, and p <0.05 was considered as statistically significant . To determine the association of various epidemiological characteristics of the patients to rif - resistant tb, odds ratio (or) and 95% confidence interval (ci) were calculated using spss statistical software version 20.0.0 (spss inc ., the relationship of smear positivity grade with the relative bacterial burden and xpert mtb / rif ct value (given in genexpert operator's manual by cepheid) was also analyzed . Out of 1612 specimens tested, 1308 were positive for mtb by xpert mtb / rif assay . There were 34 errors and 18 specimens showed no results . All these specimens were retested, and valid results were obtained except in two (one showed error in the second sample also and another could not be processed due to insufficient material in the second specimen). Zn staining of the 1308 specimens positive for mtb by xpert mtb / rif assay showed that 1240 were positive for acid fast - bacilli (afb) with 272 (21.9%) being grade 3 positive . Correlation of smear positivity grade with relative bacterial burden and ct value showed that out of total 272 grade 3 positive specimens, 69.4%, 22.5%, 6.5%, and 1.6% had high (ct <16), medium (ct 1622), low (ct 2028), and very low (ct> 28) bacterial load, respectively . Of the specimens showing grade 2, grade 1 scanty positivity, the maximum belonged to high and medium, high and medium, and medium bacterial load groups, respectively . In sputum negative specimens (68/1308) the maximum number belonged to low bacterial load group . Of 1308 mtb - positive samples by xpert tb / rif assay, 130 (9.9%) demonstrated rif resistance . Results were obtained in 4 specimens and on retesting 2 came out to be rif sensitive . All these 4 specimens were smear - positive and had ct value was> 28 . The study of the association of epidemiological characteristics of the patients to the rif - resistant tb showed that rif resistance was present in statistically significant higher number in previously treated patients in comparison to the new patients (95% ci 1.1487.135, p = 0.006 and or = 2.86) and in patients with afb - positive sputum smears to afb - negative smears (95% ci 0.91115.5, p = 0.048 and or 3.7). However, the difference was found to be statistically insignificant between the different age groups of patients (p = 0.053), male and female patients (95% ci 0.7061.57, p = 0.798 and or 1.054), patients from rural and urban background (95% ci 0.691.49, p = 0.918 and or 1.02) and hiv - positive and hiv - negative patients (95% ci 0.2194.13, p = 0.949 and or 0.953). The study of mutation pattern of rif resistance in 130 specimens revealed that in 129, rpob gene mutations in 81 bp rrdr of mtb were detected by one of the 5 different probes (a, b, c, d, e), while in one specimen, mutation combination, i.e. Mutations associated with more than one probe (a and b both) was present . The probe frequencies associated with the observed rif resistance were as follow: e 73/130 (56%), b 28/130 (21.5%), d 18/130 (13.8%), a 11/130 (8.4%), and c 1/130 (0.7%) [table 1]. Accordingly, the frequencies of mutations at 5 different rpob gene regions were 529533 (56%), 512518 (21.5%), 523529 (13.8%), 507511 (8.4%), and 518523 (0.7%). District - wise pattern of mutations seen in 130 rifampicin resistant tuberculosis specimens correlation of mutations with the rif - resistant sputum specimens obtained from different districts [table 1] revealed that probe e which represented mutation in region 529533 was the most common in all the six districts of the study . Only one specimen revealed rif resistance by probe c depicting mutation in codon region 518523 . Out of 1612 specimens tested, 1308 were positive for mtb by xpert mtb / rif assay . There were 34 errors and 18 specimens showed no results . All these specimens were retested, and valid results were obtained except in two (one showed error in the second sample also and another could not be processed due to insufficient material in the second specimen). Zn staining of the 1308 specimens positive for mtb by xpert mtb / rif assay showed that 1240 were positive for acid fast - bacilli (afb) with 272 (21.9%) being grade 3 positive . Correlation of smear positivity grade with relative bacterial burden and ct value showed that out of total 272 grade 3 positive specimens, 69.4%, 22.5%, 6.5%, and 1.6% had high (ct <16), medium (ct 1622), low (ct 2028), and very low (ct> 28) bacterial load, respectively . Of the specimens showing grade 2, grade 1 scanty positivity, the maximum belonged to high and medium, high and medium, and medium bacterial load groups, respectively . In sputum negative specimens (68/1308) the maximum number belonged to low bacterial load group . Of 1308 mtb - positive samples by xpert tb / rif assay, 130 (9.9%) demonstrated rif resistance . Results were obtained in 4 specimens and on retesting 2 came out to be rif sensitive . All these 4 specimens were smear - positive and had ct value was> 28 . The study of the association of epidemiological characteristics of the patients to the rif - resistant tb showed that rif resistance was present in statistically significant higher number in previously treated patients in comparison to the new patients (95% ci 1.1487.135, p = 0.006 and or = 2.86) and in patients with afb - positive sputum smears to afb - negative smears (95% ci 0.91115.5, p = 0.048 and or 3.7). However, the difference was found to be statistically insignificant between the different age groups of patients (p = 0.053), male and female patients (95% ci 0.7061.57, p = 0.798 and or 1.054), patients from rural and urban background (95% ci 0.691.49, p = 0.918 and or 1.02) and hiv - positive and hiv - negative patients (95% ci 0.2194.13, p = 0.949 and or 0.953). The study of mutation pattern of rif resistance in 130 specimens revealed that in 129, rpob gene mutations in 81 bp rrdr of mtb were detected by one of the 5 different probes (a, b, c, d, e), while in one specimen, mutation combination, i.e. Mutations associated with more than one probe (a and b both) was present . The probe frequencies associated with the observed rif resistance were as follow: e 73/130 (56%), b 28/130 (21.5%), d 18/130 (13.8%), a 11/130 (8.4%), and c 1/130 (0.7%) [table 1]. Accordingly, the frequencies of mutations at 5 different rpob gene regions were 529533 (56%), 512518 (21.5%), 523529 (13.8%), 507511 (8.4%), and 518523 (0.7%). District - wise pattern of mutations seen in 130 rifampicin resistant tuberculosis specimens correlation of mutations with the rif - resistant sputum specimens obtained from different districts [table 1] revealed that probe e which represented mutation in region 529533 was the most common in all the six districts of the study . Only one specimen revealed rif resistance by probe c depicting mutation in codon region 518523 . Although, india ranks first out of the 22 countries with the highest burden of tb, but the reliable information on the magnitude of mdr - tb in the country is largely unavailable . By using xpert mtb / rif assay, we observed rif resistance which is a surrogate marker of mdr - tb in 9.9% of suspected cases of mdr - tb of malwa region of punjab . However, higher prevalence of mdr - tb has been reported in other indian studies (lucknow 27.8%, new delhi 17.9%, and central india 17%). Globally, 3.7% of new cases and 20% of previously treated cases are estimated to have mdr - tb . In india, the estimated figure in new cases with mdr - tb is 2.1% with ci 1.52.7 and estimated percentage in previously treated cases is 15% with ci of 1317, respectively . Rntcp carried out drug - resistant surveys in accordance with global guidelines and indicated a low prevalence of mdr - tb, i.e.,> 3% among new cases and 1217% in previously treated cases in gujarat, and maharashtra and andhra pradesh . In the present study, the figure was 3.9% (5/126) in the new and 10.6% (125/1182) in the retreatment cases, and the difference between the two was statistically significant (p = 0.006). Gupta et al . Had also observed that mdr - tb was significantly higher in previously treated cases compared to new cases and concluded that the previous exposure to anti - tb agents was the most common cause of developing mdr . In the present study, another statistically significant association was seen between smear - positive mdr cases in comparison to smear - negative mdr cases (p = 0.048). Gupta et al . Had also observed a higher but statistically insignificant association of mdr - tb with smear positivity . This could be because all the mdr - tb patients of our study had pulmonary tb and these patients are more likely to have cavitary lesions and positive sputum smear results . Our study population showed slightly higher prevalence of rif resistance in hiv - negative patients than hiv - positive patients but statistically the difference was insignificant (p = 0.949). This is in contrast to the study of gupta et al . Who observed a statistically significant difference in mdr - tb in hiv - negative in comparison to a hiv - positive group of patients . Quy et al . Reported that the failure of treatment of mdr - tb was associated with mdr and not with hiv infection . Although statistically there were insignificant differences between different age groups showing mdr - tb (p = 0.053), a maximum number of cases in the present study were in the age group of 2140 years . Other indian studies have also reported the predominance of younger age group with mdr - tb . There were also a higher number of male patients and patients from a rural background with mdr - tb in our study . As young adult males are economically productive segment of our society, high mdr - tb in this group has several socioeconomic implications . While detecting tb / mdr cases, xpert mtb / rif assay showed 2.1% (34/1612) errors in the present study . Mboowa et al . Have reported 12% errors using xpert mtb / rif assay g3 version 3 and g4 version 5, with more than half being contributed by the former version . The lower percentage of errors in the present study could be because that we used the upgraded g4 version 5 of xpert mtb / rif assay . Similar to our study, rufai et al . Have reported 1.8% errors using xpert mtb / rif assay g4 . Nwokoye et al . Have stated that low bacterial load limits the ability of the xpert mtb / rif assay to correctly identify mutated and wildtype sequences in the core region of the rpob gene . The indeterminate results of rif resistance detected in four samples of the present study could be because of their high ct value (> 28) corresponding to the very low bacterial burden . While using xpert mtb / rif assay for the detection of mtb and rif resistance in mdr suspected cases, we could collect additional information on mutations associated with rif resistance . It was observed that the pattern of mutations in the 81 bp rrdr of mtb isolates of the present study was almost similar to that reported from other parts of india . The most common rrdr rpob gene mutations were in the gene region 529533 (56%; table 1) in all the six districts of malwa region and were recognized by probe e. mboowa et al . Too used xpert mtb / rif assay and the most common gene mutations observed were in codon 531 (58%) followed by 513 (25%), 526 (8%), and 511 (8%) designed by probes e, b, d, and a. singhal et al . Used genotype mtbdr plus assay and reported 531 as the most commonly mutated codon in 59.0% . Similarly, mani et al . Reported that the codons most commonly involved in these mutations were 531 (53%) and 526 (19%) in a study from south india . The resistant mutants isolated more frequently in clinical practice have higher mean relative fitness and their prevalence depend on their ability to survive . This might be the reason for the higher occurrence of mutations in codon 531533 . In our study, we observed rif resistance by probe c (518523) in one of the 130 rif resistant sputum samples . This is similar to the findings of gupta et al . Who used dna sequencing combined with ms - pcr assay to observe mutation in this rpob region . In contrast, no rif resistance was found to be associated with probe c in the study of mboowa et al . This could probably be because of lesser susceptibility of this genetic region to mutations or because the selection pressure shaping (producing) probe c associated rif resistance is less in malwa region of punjab (north india). Mutation combination (probe a and b) was observed in one (1/130) of the rif - resistant specimens in the present study . While, singhal et al . Found 6 strains (6/366) with more than one mutations and mboowa et al . Reported no specimen with more than one probe failure (mutation combination) while employing xpert mtb / rif assay in their study . Probably, mutations continue to arise due to the ability of mtb to adapt to drug exposure . The limitation of the study was that no gold standard was used for the comparison of xpert mtb / rif assay results . Xpert mtb / rif is a better screening tool for detection of mtb and rif resistance in a shorter period of time, and this could help improve early recognization of mdr - tb and prevention of its further transmission in malwa region of punjab . This assay also appears to be a useful technique to have simultaneous preliminary information regarding the mutation pattern of rif resistance in mtb isolates which could be helpful in understating the epidemiology of the disease and identification of hot spots for implementation of tb control program . However, for the confirmation and the detailed study on these mutations, dna sequencing remains indispensable.

The hallmark of autoimmune diseases is the pathologic activity of the immune system of an organism directed against its own cells and tissues . The disease is a direct consequence of tissue and/or organ damage as mediated by autoreactive components of the immune system, that is, autoreactive t - lymphocytes and/or autoantibodies . For diagnostic purposes, autoantibodies are the most important analytes . Within the systemic autoimmune rheumatic diseases (sard), anti - nuclear antibodies (ana), directed against various cellular components, and associated autoantibodies, such as antibodies reactive with dsdna and extractable nuclear antigens (ena), are fundamental for diagnosis [13]. Traditionally, ana are detected by indirect immunofluorescence (iif) performed on human epithelial cells (hep-2). This technique requires a multistage process consistent with visual determination of the staining pattern, serial titrations of positive sera, followed by a second test in which autoantigen specificity is determined [2, 4]. Recently, the american college of rheumatology (acr) stated that ana detection by iif is still considered the gold standard . This was primarily based on the high sensitivity of the iif assay and the inclusion of ana detection by iif assay in diagnostic criteria of systemic lupus erythematosus (sle) and autoimmune hepatitis (aih) [68]. In addition, ana can also be considered as a screening test for samples that require further testing for autoantigen specificity, that is, dsdna and ena [2, 9]. The specificity of ana detection by iif, however, is relatively poor, especially when low titres are used for screening . Indeed, at a 1: 40 serum dilution, 2530% of healthy individuals may test positive for ana and this increases even further upon ageing [1, 10]. Overall, it is recommended that the serum dilution that gives a specificity of 95% in healthy individuals should be used as cut - off . Moreover, the clinical significance rises with increasing titres [11, 12], as well as with the identification of the responsible autoantigen [1, 9]. Obviously, a positive ana test must always be interpreted cautiously and only within the clinical context of the patient . In a clinical setting where the pretest probability of sard is generally low, as in primary care, the added value of a positive ana test is lower as compared to secondary and tertiary care situations where pretest probabilities of sard are often higher . In the current study, we determined the prevalence of ana in primary (general practices), secondary (regional hospital), and tertiary care (university hospital). Besides data on ana prevalence, also ana titres and anti - ena and anti - dsdna antibodies were included in our analyses . We hypothesize that ana prevalence, ana titre, and anti - ena / dsdna reactivity increase from primary to tertiary care as these situations are expected to be also associated with an increasing pretest probability of sard . In the present study, three different patient populations from the southern part of the netherlands were evaluated and compared with each other . These three populations consisted of patients who were tested for ana between november 2011 and august 2012 in suspicion of an autoimmune disease . All ana requests were considered to involve the diagnostic workup since none of the patients had requests for ana (and/or anti - ena / dsdna) at least 4 years prior to the study period . In the first patient population (n = 1453) ana were requested by general practitioners (primary care). The second population (n = 1621) had an ana request in a regional hospital (secondary care), while the third population (n = 1168) had an ana request in a university hospital (tertiary care). Testing for ana in the first and second cohorts was performed in the atrium mc (heerlen, the netherlands), while the ana tests in the third cohort were performed in the maastricht university medical centre (mumc, maastricht, the netherlands). Furthermore, in both regional and university hospitals, the origin, that is, hospital department, of ana requests was documented . Ana detection by iif was performed on hep-2000 cells according to the instructions provided by the manufacturer (immuno concepts, sacramento, ca). Hep-2000 cells are transfected with the gene for ssa-60, which makes these cells more sensitive for ssa - antibody detection [14, 15]. Fitc - conjugated goat anti - human igg antibody was used for detection of ana . Five staining patterns were considered ana positive: homogenous, atypical speckled, speckled, centromere, and nucleolar . In case of mixed - patterns, slides were evaluated with a fluorescent microscope (axioskop, carl zeiss microscopy gmbh, jena, germany) with led light source . All slides were evaluated by two independent observers; in case of a difference in opinion, a third observer was decisive . The presence of anti - ena antibodies was screened by a commercially available line immunoassay (ana 3 profile euroline, euroimmun, lbeck, germany). After the first incubation with diluted serum, a second incubation was performed with goat anti - human igg linked to alkaline - phosphatase . Finally, a third incubation took place with bromochloroindolyl phosphate and nitro blue tetrazolium chloride (bcip / nbt) as substrate to detect anti - ena antibodies . Although the ana 3 profile euroline kit enables detection of 15 different antigens, only eight were evaluated in the current study: ro52, ss - a / ro60, ss - b / la, nrnp / sm, sm, scl-70 (topoisomerase 1), jo-1, and cenp - b . Reading of the results was automated and the colour intensities of the reactions were evaluated by the eurolinescan program (euroimmun) to enable semiquantitative determination, that is, equivocal, 1 +, 2 +, and 3 + . Results were considered positive for sm, scl-70, and jo-1 if the intensity was at least equivocal, while for ss - a / ro60, ss - b / la, nrnp / sm, and cenp - b an intensity of at least 1 + was required . Finally, the cutoff for ro52 was 2 + . Positive lia results, as defined above, were confirmed with a commercially available feia (elia, immunodiagnostics, thermo fisher scientific, freiburg, germany). This method uses highly purified (smd) or recombinant (ss - a / ro60, ro52, ss - b / la, cenp - b, scl-70, jo-1, rnp70, and u1rnp) human antigens that are coated on irradiated polystyrene cups . The assay was performed according to the manufacturer's instructions . The sera were diluted 1: 50 with dilution buffer . After binding of anti - ena antibodies, the cups were washed and subsequently incubated with mouse anti - human igg (heavy chain specific) conjugated to -galactosidase . In case of antibody association, binding was detected fluorometrically using 4-methylumbellifery--d - galactoside (0.01%) as substrate . For all antigens, values above 10 u / ml were considered positive . In primary and secondary care, anti - dsdna antibodies were detected with a commercially available feia (elia, immunodiagnostics, thermo fisher scientific). This method uses a circular plasmid dsdna, purified from escherichia coli, as antigen . The assay was performed according to the manufacturer's instructions and as described for the anti - ena antibodies . The sera were diluted 1: 10 in dilution buffer and values above 15 u / ml were considered positive . In tertiary care, anti - dsdna antibodies were detected by the crithidia luciliae immunofluorescence test (clift; immuno concepts). Fitc - conjugated goat anti - human igg antibody was used for detection of anti - dsdna antibodies . Slides were evaluated with a fluorescent microscope (axioskop, carl zeiss microscopy gmbh) with led light source . All slides were evaluated by two independent observers; in case of a difference in opinion, a third observer was decisive . All samples were tested first by ana iif . If the result was negative, no further testing was performed, unless specifically requested . However, the results of these additional tests were not included in the present study . If the ana iif was positive, irrespective of the staining pattern, titration was performed (1: 320 and 1: 1280). If a homogenous ana pattern was detected, testing for anti - dsdna antibodies was performed by feia (primary and secondary care) or clift (tertiary care). Additionally, in case of a homogenous, (atypical) speckled, or centromere pattern, typing for anti - ena antibodies was performed by lia . Because of the high correlation between the atypical speckled ana pattern and ss - a / ro60 antibodies, these antibodies were considered positive if the atypical speckled ana pattern was observed in combination with either a positive lia or a positive feia . Similarly, anti - cenp - b antibodies were considered positive when a centromere ana pattern was observed in combination with a positive anti - cenp - b result in at least one of both anti - ena antibody test systems . Since the lia does not enable specific distinction of anti - rnp antibodies, positivity for the nrnp / sm complex was followed by testing for antibodies against rnp70 and u - rnp by feia . Chicago, il) or graphpad prism version 6 (graphpad software inc ., la jolla, ca). The kolmogorov - smirnov analysis was performed to determine whether the age distributions of the three study populations were normally distributed . Furthermore, the chi square test, with yates' correction if appropriate, was performed when comparing proportions of groups . In the case of small samples, of the three patient populations included, 90 (6.2%), 175 (10.8%), and 187 (16.0%) patients from primary (n = 1453), secondary (n = 1621), and tertiary care (n = 1168), respectively, were tested positive for ana and were therefore eligible for the current study . Gender (f / m) and age (median and range) distribution were as follows: 78/12 and 57.2 (1595) for primary care, 129/46 and 57.0 (1793) for secondary care, and 130/57 and 57.3 (384) for tertiary care . The gender distribution differed significantly (p = 0.009) due to a strong female preponderance in primary care . The age distribution differed significantly (p = 0.005) due to the fact that in tertiary care age distribution was skewed negatively . Evaluation of the origin, that is, hospital departments, of the ana requests in secondary and tertiary care revealed four departments, that is, rheumatology, dermatology, internal medicine, and neurology, which requested the majority of the ana screening tests (figure 1(a)), that is, 86% and 68%, respectively . The prevalence of ana in the 3 distinct clinical settings is depicted in figure 2(a). The higher relative ana prevalence in tertiary care (16.0%) versus secondary care (10.8%) was apparent in all 4 clinical disciplines that requested the majority of ana screening test (figure 1(a)); this also holds for the other clinical disciplines (data only shown as pooled results). Typically, in secondary care 39.5% of overall ana requests came from the rheumatology department, while in tertiary care this was 15.4% . Within the positive ana cohorts, patients from primary care had relatively low titres out of 90 positive ana tests 63 sera (70%) revealed an ana titre of 1: 80 . In secondary and tertiary care, a titre of 1: consequently, higher titres were observed more frequently in secondary and tertiary care than in primary care . The distribution in titres between secondary and tertiary care was not different . At first glance, there is no apparent difference in the distribution of ana patterns between the three health care levels (figure 2(c)). Also, comparison of the distribution of ana patterns with a titre of 1: 1280, considered to have the highest positive likelihood ratio, showed no significant differences (data not shown). The prevalence of anti - ena and anti - dsdna antibodies, as defined by the algorithm described above, is presented in figure 3(a). In the primary care, 19 (21.1%) patients with positive ana (n = 90) were tested positive for anti - ena and/or anti - dsdna antibodies, that is, 1.3% of the total cohort . In the secondary and tertiary care, 68 of 175 (38.9%) and 40 of 187 (21.4%) ana positive patients revealed anti - ena and/or anti - dsdna reactivity, respectively . This is 4.2% and 3.4% of the total secondary and tertiary care cohorts, respectively (p = 0.367). Significantly more positive anti - ena and/or anti - dsdna results were found in the total secondary and tertiary care cohorts than in the primary care cohort (p <0.0001 and p = 0.0006, resp . ). In secondary care, the prevalence of anti - ena and/or anti - dsdna reactivity within the positive ana samples was the highest in the requests from rheumatology (figure 1(b)). In tertiary care, however, departments of rheumatology and neurology had, on average, reduced prevalence of anti - ena and/or anti - dsdna reactivity as compared to the departments of dermatology and internal medicine (figure 1(b)). Since the relevance of anti - ena and anti - dsdna antibodies is considered to increase when combined reactivity is observed, we analysed the prevalence of anti - ena and/or anti - dsdna antibodies in relation to combined reactivity (figure 3(b)). In the primary care setting, single positivity appeared to be more abundant than in secondary and tertiary care, but this difference was statistically not significant . No differences were observed between secondary and tertiary care settings either . With respect to the antigens recognized by the specific antibodies, no apparent differences were observed in the prevalence of antibodies reactive to rnp, ssa60, ro52, ssb, cenp - b, and dsdna (figure 3(c)). Anti - scl70 antibodies were only detected in samples of patients from secondary and tertiary care . However, the absolute number, one in each cohort, was low in both of these settings . In the present study on the analyses of ana prevalence, ana titre and anti - ena specificity in the primary, secondary, and tertiary care, our results indicate that (i) ana prevalence significantly increases from primary to tertiary care, (ii) low titres (1: 80) are more frequently observed in the primary care, and (iii) anti - ena and anti - dsdna specificities are significantly more prevalent in the secondary care than in the primary care . Interpretation of the data obtained in the current study is highly dependent on the viewpoint on clinical utility of an ana test result . This result may help the clinician to identify a patient with sard, but especially in situations with low pretest probabilities of such diseases, the risk of false interpretation of a positive ana is high . This risk of false positive interpretation will decrease if the positive ana is characterized by high titre and includes (multi)-reactivity for ena and/or dsdna, since these characteristics are associated with higher positive likelihood ratios [11, 12, 17]. Next, one has to realize that ana testing is performed in the context of multiple diseases, varying from distinct sard to autoimmune liver diseases . Interpretation of a positive ana test may be different for each distinct disease: patients with, for instance, aih or systemic sclerosis often have a positive ana with no ena reactivity, while a positive ana test as such is part of the classification criteria for sle, as well as aih [68]. A negative ana test result, on the other hand, may also be very useful to exclude a specific set of diseases and may drive attention to other diseases . Again, this differs for the distinct sard: a negative ana test has a high negative predictive value for sle and systemic sclerosis but is less helpful to exclude sjgren's syndrome or myositis [2, 3]. Obviously, definite interpretation of our dataset is hampered by the lack of clinical data from the patients of the three cohorts . The assumption of the primary care having a relatively low pretest probability was based on previous studies [13, 18, 19]. For future studies, we recommend the inclusion of clinical data in order to be able to thoroughly assess pretest probabilities and strengthen our assumptions . This study, however, also has noteworthy strengths, that is, only data obtained during the diagnostic workup of patients (not follow - up samples) were included and the same testing algorithm and reagents (except for detection of anti - dsdna antibodies) were used for all three patient cohorts . As expected, we observed a gradual increase in the prevalence of ana from primary to tertiary care . In our present study ana was detected by iif in a screening dilution of 1: 80 . In several studies it has been reported that 1015% of healthy controls are ana positive in this serum dilution [1, 10, 20]. Obviously, a positive ana test result is not only dependent on the dilution factor but also on the quality of other reagents, that is, fluorescent conjugate, cell substrate, and the fluorescent microscope . For standardization purposes, it has, therefore, been recommended to screen for ana with a specificity of 95% . Taking this into account, the mere presence of ana in our primary care cohort (prevalence 6.2%) lacks discriminating power to identify patients with sard . On the other hand, due to the high negative predictive value of a negative ana result furthermore, since both high titre and anti - ena and/or anti - dsdna (multi-)reactivity may increase the likelihood of identifying a patient with sard [11, 12, 17], it is apparent in our primary care cohort that 30% of ana positive sera are of medium to high titre (n = 27), 21% contain anti - ena and/or anti - dsdna reactivity (n = 19), and 37% of the latter reveal multireactivity (n = 7). In our secondary and tertiary cohorts, the ana prevalence is also relatively low (11 and 16%, resp . ), but in both clinical settings about half of the ana positive sera are of high titre (48 - 49%). As expected, the observed anti - ena and/or anti - dsdna reactivity of ana positive sera in the secondary care (39%) is higher than in the primary care . Surprisingly, anti - ena and/or anti - dsdna reactivity in the tertiary care (21%) is lower than in the secondary care and similar to the primary care . This might be related to the spectrum of diseases investigated in the tertiary versus the secondary centres . Also the higher number of ana requests by tertiary care departments not typically involved in the diagnosis of sard might be the result of academic profiling of the respective departments . For instance, the cardiology department of the mumc is specialized in inflammatory cardiomyopathies and a possible autoimmune aetiology of these diseases . Cardiology requested 108 ana tests (9.2%), revealing 13% ana positive results of which 20% was anti - ena and/or anti - dsdna antibody positive . A second important difference is the presence of a division of clinical immunology within the department of internal medicine of the tertiary care hospital, implying that many patients with sard are evaluated by clinical immunologists instead of rheumatologists . The latter difference might at least explain the lower relative prevalence of anti - ena and anti - dsdna antibodies in the rheumatology department of the tertiary care hospital . Our study shows that in secondary care the majority of positive ana results (92.6%), as well as the positive anti - ena and anti - dsdna results (95.6%), are linked to the departments of rheumatology, dermatology, internal medicine, and neurology . Obviously, patients with the highest pretest probabilities for sard, as associated with the initial clinical presentation, are most likely referred to these clinical departments . In clinical settings with lower pretest probabilities, ana positive sera are more likely to be of no clinical significance [13, 18, 19]. It might therefore be an option, in particular in primary care settings, to move away from the traditional ana screening test . In this context, the recent discovery of the dense fine speckled (dfs70) antigen is promising and could offer a possible solution for the identification and exclusion of positive sera with no clinical relevance [21, 22]. The typical ana dense fine speckled pattern (dfs), known to be associated with the dfs70 antigen, has been found to be commonly prevalent in healthy individuals with ana positive sera (33.1%), whereas in sard patients 0.0% of the sera revealed a dfs pattern . Another study revealed that 2 - 3% of sard patients had antibodies directed to the dfs70 antigen . Obviously, in sard patients other anti - ena antibodies might be present that hamper correct recognition of the dfs iif pattern indeed, the identification and correct interpretation of the dfs pattern might prove to be challenging for diagnostic laboratories and would require additional training . Moreover, the majority of patterns recognized do not seem to be compatible with a dfs pattern, but this might be the consequence of only interpreting the strongest pattern in the current study . Since the identification of the dfs pattern might be challenging for routine diagnostic laboratories and inaccurate interpretation can have significant consequences, an immunoadsorption protocol to diminish anti - dfs70 antibody reactivity to hep-2000 cells could be implemented in the current iif assay in order to significantly improve the performance characteristics of the ana iif test [21, 25]. This approach sustains recognition of sard - related autoantibodies in sera with combined reactivities, that is, anti - dfs70 and other anti - ena antibodies . Another alternative testing algorithm could, instead of ana iif, include solid phase assays including multiplex and screening assays for well - defined anti - ena and anti - dsdna reactive antibodies [11, 12, 26, 27]. Both approaches enable to distinguish or reduce the number of positive ana results lacking clinical relevance . In addition, multiplex anti - ena screening assays are considered to better recognize particular antigens, for example, ssa and jo-1, as compared to ana iif testing [3, 11, 12, 28]. Altogether, the results indicate that in the primary care the usage of traditional ana screening tests is more prone to false - interpretation of positive ana results . Rather, an alternative testing algorithm for detection of patients with sard might be more appropriate . This might either be achieved by immunoadsorption of anti - dfs70 antibodies or direct screening for anti - ena antibodies . Obviously, a patient with severe clinical manifestations typical for sard presenting in a primary care setting should be referred directly, that is, without any laboratory testing, to the rheumatologist or clinical immunologist . This recommendation may not only apply for general practitioners but may also hold for clinical departments that are less likely to encounter patients suspected of sard.

Mdct benefits from having a high spatial resolution and fast acquisition time, making it an ideal tool to stage and restage tumours . The routine use of intravenous (and oral contrast) media, unless contraindicated, also helps to delineate sites of disease, especially within solid organs . The identification of nodal involvement on ct is based on nodal size, typically using a minimum cutoff of 1 cm in short axis diameter, although this can lead to both under- and over - staging of metastatic nodal involvement . The purpose of this pictorial review is to discuss the imaging appearances on ct of some of the more common cancers arising within the abdomen and pelvis and to describe their typical sites of local, nodal and haematogenous tumour spread . Cancers arising from the stomach, pancreas, colon, kidney, ovary and prostate will be reviewed, with summary tables provided . Ninety percent of tumours are adenocarcinomas, which are subdivided into two main histological types: (1) the well - differentiated (intestinal) type, which is associated with atrophic gastritis, occurs in older patients and has a better survival rate and (2) the undifferentiated (diffuse / signet ring) type, which is more common, occurs more frequently in women and has a poorer prognosis . Gastric adenocarcinoma occurs either proximally (cardia) or distally (non - cardia), with the former increasing in incidence whilst the latter is declining (see table 1). Table 1summary table of local, nodal and haematogenous spread in gastric cancerlocal spreadlocal spread into adjacent structures (e.g. Pancreas, colon, spleen)lymph node spreadperigastric: pericardial; lesser curvature; greater curvature; suprapyloricextraperigastric: left gastric; common hepatic; coeliac; splenic hilum and artery; hepatic pedicle; retropancreatic; mesenteric root; middle colic; para - aortichaematogenous spreadmost commonly the liver (25% at presentation) and peritoneumnotestranscoelomic spread can occur through the peritoneum (e.g. Kruckenberg tumours)retropancreatic, para - aortic and mesenteric nodes are classified as m1 metastatic disease nodal disease in gist tumours is extremely rare summary table of local, nodal and haematogenous spread in gastric cancer on ct, the use of oral contrast agents to distend the stomach is essential, preferably with water rather than dilute gastrograffin to avoid beam - hardening artefacts . Involvement of the stomach wall is seen as thickening or irregularity of the inner / middle layer of the gastric wall (t1 tumours) or transmural thickening with abnormal contrast enhancement (t2 tumours). Wall thickness has been reported to be greater and of higher attenuation in the diffuse histological type compared to the intestinal type . Gastric cancer spreads in a predictable fashion through the gastric wall with the presence of perigastric fat stranding and nodular outline suggestive of serosal involvement (t3 tumours) (fig . 1). 1gastric adenocarcinoma: axial contrast - enhanced ct (cect) showing tumour arising from the lesser curvature of the stomach (asterisk) associated with enlarged regional gastrohepatic nodes (arrows) gastric adenocarcinoma: axial contrast - enhanced ct (cect) showing tumour arising from the lesser curvature of the stomach (asterisk) associated with enlarged regional gastrohepatic nodes (arrows) spread to perigastric and less frequently other intra - abdominal nodes may occur (table 1; fig . 1). Sign describes the clinical finding of an enlarged left supraclavicular lymph node due to metastatic involvement . Abdominal cancers may metastasise to this site via the thoracic duct, and gastric malignancy is classically described as the commonest primary tumour to do this . Obstructive jaundice with associated bile duct dilatation may occur, due to enlarged porta hepatis nodes or due to the spread of gastric tumour via the gastrohepatic ligament directly into the liver . Haematogeneous spread via the portal vein to the liver occurs in 25% of patients at presentation, with liver metastases typically appearing as rim - enhancing low - attenuation lesions during the portal venous phase . Peritoneal disease secondary to gastric adenocarcinoma may mimic the appearance of peritoneal disease secondary to metastatic ovarian carcinoma, with omental cake and/or discrete peritoneal deposits noted within the abdominal cavity . Trans - coelomic spread is also characteristic of gastric cancer, with spread to the ovary resulting in a krukenberg tumour that typically appears as a mixed solid / cystic adnexal mass . Involvement of the peritoneal reflection within the pelvis can result in a positive blumer s shelf finding, with tumour found high on the anterior rectal wall . It should be noted that other histological tumour types arising from the stomach have different patterns of tumour spread . Gastrointestinal stromal tumours (gists) are the most common primary mesenchymal neoplasms of the gastrointestinal tract, distinct from true smooth muscle and neural tumours, and are derived from the interstitial cells of cajal . They are most frequently located within the stomach (6070%) and should be regarded as having malignant potential . Gists typically involve the outer muscular layer of the gi tract, and thus often demonstrate exophytic growth (fig . 2). Most appear as well - defined extraluminal or combined intraluminal / extraluminal masses on ct . If small, they tend to be of homogeneous attenuation; if larger, they are more heterogeneous on both the unenhanced and enhanced scans because of necrosis or haemorrhage . The central areas of necrosis may communicate with the gastric lumen . If arising from the stomach, the tumour may extend into the gastrohepatic ligament, the gastrosplenic ligament and the lesser sac . Nodal spread is extremely rare, but haematogenous spread can occur, often to the liver with nearly 50% of patients with gists presenting with metastases (fig . 2). 2gastrointestinal stromal tumour (gist): axial cect showing typical large low attenuation exophytic tumour (asterisk) arising from the greater curvature of the stomach associated with liver metastasis (arrow). Note there are no significantly enlarged regional nodes gastrointestinal stromal tumour (gist): axial cect showing typical large low attenuation exophytic tumour (asterisk) arising from the greater curvature of the stomach associated with liver metastasis (arrow). Note there are no significantly enlarged regional nodes pancreatic ductal adenocarcinoma accounts for more than 80% of malignant tumours of the pancreas, and ct is the initial imaging modality used for staging of suspected pancreatic cancer with ct (along with mri and endoscopic sonography) used to distinguish between potentially resectable and non - resectable patients . For cancer at the head of the pancreas, contraindications to surgery include distant metastases, circumferential involvement of the superior mesenteric vein - portal vein segment more than 2 cm long, thrombus in the vein, and occlusion or circumferential invasion of the celiac, hepatic or superior mesenteric arteries (see table 2). Table 2summary table of local, nodal and haematogenous spread in pancreatic cancerlocal spreadtumour spreads by direct perivascular and perineural invasion;local invasion can involve the stomach, duodenum and retroperitoneum;head / uncinate process tumours: these usually extend along the sma and mesenteric root;body / tail tumours: these usually infiltrate the celiac, hepatic or splenic arterieslymph node spreadprimary drainage: superior, inferior, anterior, posterior and splenic lymph nodes;secondary drainage: porta hepatis, common hepatic, coeliac, mesenteric root lymph nodes;tertiary drainage: peri - aortic and distal superior mesenteric lymph nodeshaematogenous spreadthese usually involve the liver and peritoneal surfacesnoteearly lymphatic and haematogenous micrometastases at presentation are common summary table of local, nodal and haematogenous spread in pancreatic cancer when performing a staging ct, ideally at least two contrast - enhanced acquisitions (during the late arterial and venous phases) are advised . The pancreas is a retroperitoneal organ and has a close anatomic relationship with the abdominal peritoneal reflections . The pancreatic head is connected to the liver and lesser curvature of the stomach via the hepatoduodenal ligament and gastrohepatic ligaments, respectively . The tail of the pancreas is in continuity with the hilum of the spleen and the greater curvature of the stomach via the splenorenal and gastrosplenic ligaments . Pancreatic cancer can invade these adjacent peritoneal ligaments (as well as the transverse mesocolon) to involve the stomach, duodenum (fig . Tumour tends to spread by direct perivascular and perineural invasion within the subperitoneal space . If arising from the pancreatic head or uncinate process, tumours usually extend along the superior mesenteric artery and root of the mesentery . Biliary and pancreatic duct dilatation 3pancreatic adenocarcinoma: a axial cect showing an example of a tumour arising in the head of the pancreas (asterisk) with invasion into the duodenum . There is a focal thrombus in the portal vein (arrow); b axial cect in same patient as (a); there is associated intra- (arrows) and extrahepatic (not shown) bile duct dilatation; c axial cect showing a large bulky tumour in a different patient arising in the body of the pancreas (asterisk) encasing the origin of the celiac axis pancreatic adenocarcinoma: a axial cect showing an example of a tumour arising in the head of the pancreas (asterisk) with invasion into the duodenum . There is a focal thrombus in the portal vein (arrow); b axial cect in same patient as (a); there is associated intra- (arrows) and extrahepatic (not shown) bile duct dilatation; c axial cect showing a large bulky tumour in a different patient arising in the body of the pancreas (asterisk) encasing the origin of the celiac axis spread to regional nodes occurs, involving either peripancreatic nodes, nodes at the celiac axis and porta hepatis, or sometimes nodes further afield (table 2). Most colorectal cancers arise from adenomatous polyps, with polyps greater than 2 cm in size associated with a greater than 40% risk of malignancy . For local staging of rectal cancer, mri is now the standard imaging modality because of its increased soft tissue resolution and multiplanar capabilities . Colorectal tumours may appear as polypoid or infiltrative lesions of the bowel wall (fig . Early colonic tumours are better detected when endoluminal distension is given and the colon has been prepared, as in ct colonography . Tumour spread through the serosa appears as pericolonic fat stranding, but may be confused with an associated desmoplastic reaction (possibly mimicking diverticulitis within the sigmoid colon). The presence of fluid in the root of the sigmoid mesentery and engorgement of the adjacent sigmoid mesenteric vasculature favours diverticulitis, whilst the presence of pericolic lymph nodes is suspicious for colon cancer . Tumour spread to adjacent organs is suggested by loss of the fat planes and direct invasion (see table 3). Table 3summary table of local, nodal and haematogenous spread in colorectal cancerlocal spreadinvasion through the bowel wall into the peri - colonic fat and adjacent structureslymph node spreadfollows the vascular distribution of vessels in mesocolon;ascending mesocolon: nodes along the ileocolic vessels and right colic vessel;transverse mesocolon: nodes along the middle colic vessels;sigmoid and descending mesocolon: nodes along the inferior mesenteric vein;regional lymph nodes for rectal cancers include: mesorectal, sigmoid mesenteric, inferior mesenteric, lateral sacral, presacral, internal iliac, sacral promontory, superior rectal, middle rectal and inferior rectalhaematogenous spreadliver (via the portal vein)> lung, adrenal glands, bonesnotesin rectal cancers that have perforated the peritoneum, transcoelomic spread favours the lower right small bowel mesentery and the pouch of douglasfig . 4colonic adenocarcinoma: a axial non - contrast - enhanced ct showing tumour (asterisk) in the ascending colon with enlarged ileocolic nodes (arrows); b axial contrast - enhanced ct shows a large liver metastasis (asterisk) summary table of local, nodal and haematogenous spread in colorectal cancer colonic adenocarcinoma: a axial non - contrast - enhanced ct showing tumour (asterisk) in the ascending colon with enlarged ileocolic nodes (arrows); b axial contrast - enhanced ct shows a large liver metastasis (asterisk) nodal spread depends on the site of the primary tumour and follows the vascular distribution of the vessels within the mesocolon [11, 12]. These vessels include the ileocolic vessels and right colic vessel for the ascending mesocolon, the middle colic vessels for the transverse mesocolon, and the inferior mesenteric vein for the sigmoid and descending mesocolon . For rectal tumours, drainage is usually cranial within the mesorectum to involve the regional lymph nodes (table 3). 5) is less common, with involvement of the inguinal nodes only seen with lower rectal tumours when there is proximal lymphatic blockage (e.g. Extensive adenopathy). 5rectal adenocarcinoma: coronal reformat cect showing a bulky rectal tumour (asterisk) with enlarged right internal iliac nodes (double asterisk) and left paraaortic node (arrow) rectal adenocarcinoma: coronal reformat cect showing a bulky rectal tumour (asterisk) with enlarged right internal iliac nodes (double asterisk) and left paraaortic node (arrow) metastatic disease at presentation is uncommon (<1015% of cases). Haematogeneous spread is predominantly seen within the liver, with metastases deriving their blood supply from the hepatic artery (compared to normal liver parenchyma, which is primarily supplied by the portal vein). As a result, liver metastases imaged during the portal venous phase are seen as heterogeneous ring - enhancing metastases that are hypodense to the surrounding liver parenchyma (fig . For example, the venous drainage of the colon and upper rectum is via the portal vein, and thus the liver is a common site of spread . However, the lower rectum has a dual drainage, with the superior haemorrhoidal vein draining into the inferior mesenteric vein and then into the portal vein, and the middle and inferior haemorrhoidal veins draining into the pelvic veins and then directly into the inferior vena cava . This explains why distal rectal cancers can result in isolated pulmonary metastases without hepatic metastases . If rectal tumours perforate the peritoneal membrane, transcoelomic spread may occur, favouring the lower right small bowel mesentery and pouch of douglas . If colorectal tumours are of mucinous histology, widespread intraperitoneal mucinous metastases may occur with characteristic scalloping of adjacent viscera (pseudomyxoma peritoneii) (fig . 6pseudomyxoma peritoneii: axial cect showing mucinous ascites exerting mass effect (scalloping) on the solid organs; primary was of gastrointestinal tract origin pseudomyxoma peritoneii: axial cect showing mucinous ascites exerting mass effect (scalloping) on the solid organs; primary was of gastrointestinal tract origin on ct, renal tumours appear as hypervascular masses, with larger lesions of heterogenous attenuation . With increasing size, they spread through the renal capsule into the perinephric fat to involve gerota s fascia . Early extension through the renal capsule is recognized by an indistinct tumour margin, thickened perirenal fascia and perinephric fat stranding . Extension into the renal vein occurs in 20% of patients at presentation with involvement of the inferior vena cava (ivc) in 510% of patients (fig . Tumour thrombus is seen as a filling defect within the vein, which should not be confused with streaming artefact from unopacified blood . The presence of venous distension on its own may be misleading as rccs are hypervascular and hence tend to have increased blood flow and venous drainage . Distinction between tumour thrombus and bland thrombus within the ivc may be difficult, although the former may have enhancing tumour vessels within it . In advanced tumours, direct invasion into adjacent structures such as the diaphragm, and posterior abdominal wall muscles may be seen (see table 4). Table 4summary table of local, nodal and haematogenous spread in renal cell carcinomalocal spreadperinephric fat; ipsilateral adrenal; adjacent viscera (including muscles);renal vein invasion (ivc)lymph node spreadvia lymphatics following the renal vessels to the ipsilateral para - aortic nodes; direct connections with the thoracic duct and mediastinum also existhaematogenous spreadcommon sites: lungs> bones, cns, adrenalsnoteextension into renal vein occurs in 20% of patients at presentation; ivc involvement in 510%fig . 7metastatic renal cell carcinoma: a coronal reformat cect showing the inferior vena cava (ivc) greatly expanded by tumour thrombus (arrows) with associated enlarged mediastinal nodes (asterisks). The primary renal tumour has been excised; b axial cect showing enhancing lesion in the left gluteus maximus muscle in the same patient, which increased in size on interval ct studies, in keeping with a metastatic deposit; note the numerous venous collaterals in the anterior abdomen (short arrows) due to the ivc thrombus; c axial cect shows a large lytic deposit in the lumbar spine (asterisk) in the same patient causing compromise of the spinal canal and cord (arrows) summary table of local, nodal and haematogenous spread in renal cell carcinoma metastatic renal cell carcinoma: a coronal reformat cect showing the inferior vena cava (ivc) greatly expanded by tumour thrombus (arrows) with associated enlarged mediastinal nodes (asterisks). The primary renal tumour has been excised; b axial cect showing enhancing lesion in the left gluteus maximus muscle in the same patient, which increased in size on interval ct studies, in keeping with a metastatic deposit; note the numerous venous collaterals in the anterior abdomen (short arrows) due to the ivc thrombus; c axial cect shows a large lytic deposit in the lumbar spine (asterisk) in the same patient causing compromise of the spinal canal and cord (arrows) lymphatic spread from rcc tends to follow the renal veins to involve the ipsilateral para - aortic nodes . There are also direct connections with the thoracic duct and mediastinum, which can account for the rare presence of mediastinal and hilar node involvement (fig . Metastatic involvement in renal cell carcinoma is seen (in order of decreasing frequency) in: the lung (5060%), bone (3040%), liver (3040%), adrenal gland (5%), contralateral kidney (5%), retoperitoneum (5%) and brain (5%). More unusual sites that have been reported include the pancreas, peritoneum, bowel, thyroid and muscle (fig . 7c). Cross - sectional imaging (ct or mri) is used for preoperative tumour assessment, with ct being the imaging modality of choice for assessing metastatic disease . Bowel opacification is usually achieved with a positive oral contrast medium (e.g. Dilute gastrograffin), which is essential for the detection of peritoneal deposits . Intravenous contrast is also routinely administered unless contraindicated, with imaging performed in the portal venous phase . Conversely, local pelvic spread is better assessed with pelvic mri than ct (see table 5). Table 5summary table of local, nodal and haematogenous spread in ovarian carcinomalocal spreaduterus and broad ligament (via the fallopian tube);direct invasion of the rectum, colon, bladder and pelvic side walllymph node spreadvia lymphatics travelling along with the ovarian vessels to terminate in common iliac and para - aortic nodes;via the broad ligament to terminate in the internal iliac and obturator nodes;via the round ligament to terminate in the external iliac and inguinal nodestrans - coelomic spreadcommon sites: undersurface of the diaphragm, liver surface, pouch of douglas, omentum, serosal bowel surfaceshaematogenous spreadthis occurs late during the disease;liver> lungs, kidney, bonenoteascites arise from increased production from tumour surfaces and/or occlusion of retroperitoneal lymph nodes summary table of local, nodal and haematogenous spread in ovarian carcinoma primary ovarian cancer has variable appearances, presenting either as a solid mass or mixed solid / cystic lesion (fig . Ovarian cancer spreads locally to adnexal structures, the uterus and the contralateral ovary, although bilateral ovarian cancer may occur in 1150% of cases . Invasion of the pelvic side wall is indicated when the tumour is seen encasing the iliac vessels or is within 3 mm of the pelvic sidewall . 8ovarian carcinoma: coronal reformat cect showing a large solid / cystic adnexal mass arising from the pelvis (asterisk), with large - volume para - aortic adenopathy (double asterisk), ascites and small peritoneal deposits (arrows). Although para - aortic nodal involvement is seen in metastatic ovarian carcinoma, the large volume is rather unusual; b sagittal reformat cect showing subcapsular deposits over the liver in a different patient (arrows); c axial cect in the same patient as (b) showing a thick omental cake (arrows); d axial cect in the same patient as (b) showing multiple enlarged (short axis diameter> 5 mm) cardiophrenic nodes (arrows) ovarian carcinoma: coronal reformat cect showing a large solid / cystic adnexal mass arising from the pelvis (asterisk), with large - volume para - aortic adenopathy (double asterisk), ascites and small peritoneal deposits (arrows). Although para - aortic nodal involvement is seen in metastatic ovarian carcinoma, the large volume is rather unusual; b sagittal reformat cect showing subcapsular deposits over the liver in a different patient (arrows); c axial cect in the same patient as (b) showing a thick omental cake (arrows); d axial cect in the same patient as (b) showing multiple enlarged (short axis diameter> 5 mm) cardiophrenic nodes (arrows) peritoneal dissemination is the commonest mode of spread, being found in approximately 70% of patients at presentation . Common sites include the greater omentum, paracolic gutters, the pouch of douglas, the liver capsule (fig . Less commonly, implants may be seen within the mesentery, along the porta hepatis, lesser sac, splenic surface and gastrosplenic ligament [1618]. Ascites are often present, due to either increased production from tumour surfaces or reduced peritoneal resorption from invasion of lymphatics by tumour cells . Peritoneal deposits on ct have a variety of appearances including enhancing nodular soft tissue lesions, linear / plaque - like thickening of peritoneal reflections (fig . 8c), tiny calcifications or mixed solid and cystic or purely cystic lesions [19, 20]. The involvement of bowel serosa can lead to (asymmetric) bowel wall thickening, tethering of bowel loops and bowel obstruction . Liver surface implants (subcapsular deposits) may be seen, with deposits causing characteristic scalloping of the liver capsule . Metastatic mucinous tumours of the ovary can lead to pseudomyxoma peritoneii, although in many of these cases, the primary mucinous tumour actually arises from the gastrointestinal tract with secondary involvement of the ovary . Lymphatic spread occurs via three routes and is usually seen in conjunction with peritoneal spread . Routes include: (1) along the ovarian vessels to the common iliac and para - aortic nodes (fig . 8a); (2) via the broad ligament and parametria to the internal iliac and obturator nodes; and (3) rarely via the round ligaments to the external iliac and inguinal nodes . Cardiophrenic nodes (defined as> 5 mm short axis diameter on ct) are characteristically involved as they are the main lymphatic drainage route from the peritoneal cavity (fig . Haematogenous dissemination is rare in ovarian carcinoma with a malignant pleural effusion being the most common manifestation . At presentation, other sides of spread include the spleen, kidneys, adrenals, lungs, brain and bone . Mri is traditionally used for local staging of prostate cancer because of its high contrast resolution (as in rectal cancer). It is able to differentiate organ - confined disease from tumour that demonstrates extracapsular spread . Direct involvement of the seminal vesicles or adjacent organs is also well seen on mri . Ct is used for nodal and distant staging of prostate cancer (see table 6). Table 6summary table of local, nodal and haematogenous spread in prostate cancerlocal spreaddirect extension through the prostate capsule into the seminal vesicles and bladder baselymph node spreadorder of nodal involvement: obturator, presacral, internal iliac, common iliachaematogenous spreadbone>> lung and liver;spinal bone metastases are the commonest site (due to the direct communication between the presacral and periprostatic veins)notesdenonvillier s fascia forms a relative natural barrier to rectal spread;tumours at the apex of the prostate are more likely to demonstrate extracapsular extension because of relatively little capsule at this level summary table of local, nodal and haematogenous spread in prostate cancer classically, two lymph node metastatic patterns have been reported . 9a) and para - aortic lymph nodes, and this pelvic nodal involvement is sequential with spread to the obturator nodes, presacral nodes, internal iliac and common iliac nodes . Pattern 2 involves para - aortic nodes only, with this second pattern of nodal spread felt to be due to haematogenous dissemination . 9prostate carcinoma: a coronal reformat cect showing irregular enlarged prostate tumour (white arrow) extending into the bladder with enlarged left pelvic side wall node (black arrow), liver metastases (double arrows) and lesion in the left pubic ramus with large soft tissue component (*). Note the bones are sclerotic in keeping with diffuse bony metastases; b sagittal reformat cect in a different patient on bone windows, showing multiple sclerotic metastases in the thoracolumbar spine prostate carcinoma: a coronal reformat cect showing irregular enlarged prostate tumour (white arrow) extending into the bladder with enlarged left pelvic side wall node (black arrow), liver metastases (double arrows) and lesion in the left pubic ramus with large soft tissue component (*). Note the bones are sclerotic in keeping with diffuse bony metastases; b sagittal reformat cect in a different patient on bone windows, showing multiple sclerotic metastases in the thoracolumbar spine distant spread occurs in one third of patients with involved sites including bone (90%), lung (50%), liver (25%) (fig . Osteoblastic sclerotic metastases rather than lytic bone lesions are usually seen on ct . In order of decreasing frequency, skeletal metastases are seen within the vertebrae (fig . Spinal involvement is initially more common within the lumbar region and less likely within the cervical spine (97% cf . Pulmonary metastases occur in between 527% of patients at presentation and are more often seen in association with the second pattern of nodal involvement . Pulmonary involvement can either appear as lymphangitis carcinomatosa (due to direct invasion of the pulmonary lymphatics) or as pulmonary nodules (due to haematogenous spread) [22, 24]. Ninety percent of tumours are adenocarcinomas, which are subdivided into two main histological types: (1) the well - differentiated (intestinal) type, which is associated with atrophic gastritis, occurs in older patients and has a better survival rate and (2) the undifferentiated (diffuse / signet ring) type, which is more common, occurs more frequently in women and has a poorer prognosis . Gastric adenocarcinoma occurs either proximally (cardia) or distally (non - cardia), with the former increasing in incidence whilst the latter is declining (see table 1). Table 1summary table of local, nodal and haematogenous spread in gastric cancerlocal spreadlocal spread into adjacent structures (e.g. Pancreas, colon, spleen)lymph node spreadperigastric: pericardial; lesser curvature; greater curvature; suprapyloricextraperigastric: left gastric; common hepatic; coeliac; splenic hilum and artery; hepatic pedicle; retropancreatic; mesenteric root; middle colic; para - aortichaematogenous spreadmost commonly the liver (25% at presentation) and peritoneumnotestranscoelomic spread can occur through the peritoneum (e.g. Kruckenberg tumours)retropancreatic, para - aortic and mesenteric nodes are classified as m1 metastatic disease nodal disease in gist tumours is extremely rare summary table of local, nodal and haematogenous spread in gastric cancer on ct, the use of oral contrast agents to distend the stomach is essential, preferably with water rather than dilute gastrograffin to avoid beam - hardening artefacts . Involvement of the stomach wall is seen as thickening or irregularity of the inner / middle layer of the gastric wall (t1 tumours) or transmural thickening with abnormal contrast enhancement (t2 tumours). Wall thickness has been reported to be greater and of higher attenuation in the diffuse histological type compared to the intestinal type . Gastric cancer spreads in a predictable fashion through the gastric wall with the presence of perigastric fat stranding and nodular outline suggestive of serosal involvement (t3 tumours) (fig . 1). 1gastric adenocarcinoma: axial contrast - enhanced ct (cect) showing tumour arising from the lesser curvature of the stomach (asterisk) associated with enlarged regional gastrohepatic nodes (arrows) gastric adenocarcinoma: axial contrast - enhanced ct (cect) showing tumour arising from the lesser curvature of the stomach (asterisk) associated with enlarged regional gastrohepatic nodes (arrows) spread to perigastric and less frequently other intra - abdominal nodes may occur (table 1; fig . 1). Sign describes the clinical finding of an enlarged left supraclavicular lymph node due to metastatic involvement . Abdominal cancers may metastasise to this site via the thoracic duct, and gastric malignancy is classically described as the commonest primary tumour to do this . Obstructive jaundice with associated bile duct dilatation may occur, due to enlarged porta hepatis nodes or due to the spread of gastric tumour via the gastrohepatic ligament directly into the liver . Haematogeneous spread via the portal vein to the liver occurs in 25% of patients at presentation, with liver metastases typically appearing as rim - enhancing low - attenuation lesions during the portal venous phase . Peritoneal disease secondary to gastric adenocarcinoma may mimic the appearance of peritoneal disease secondary to metastatic ovarian carcinoma, with omental cake and/or discrete peritoneal deposits noted within the abdominal cavity . Trans - coelomic spread is also characteristic of gastric cancer, with spread to the ovary resulting in a krukenberg tumour that typically appears as a mixed solid / cystic adnexal mass . Involvement of the peritoneal reflection within the pelvis can result in a positive blumer s shelf finding, with tumour found high on the anterior rectal wall . It should be noted that other histological tumour types arising from the stomach have different patterns of tumour spread . Gastrointestinal stromal tumours (gists) are the most common primary mesenchymal neoplasms of the gastrointestinal tract, distinct from true smooth muscle and neural tumours, and are derived from the interstitial cells of cajal . They are most frequently located within the stomach (6070%) and should be regarded as having malignant potential . Gists typically involve the outer muscular layer of the gi tract, and thus often demonstrate exophytic growth (fig . 2). Most appear as well - defined extraluminal or combined intraluminal / extraluminal masses on ct . If small, they tend to be of homogeneous attenuation; if larger, they are more heterogeneous on both the unenhanced and enhanced scans because of necrosis or haemorrhage . The central areas of necrosis may communicate with the gastric lumen . If arising from the stomach, the tumour may extend into the gastrohepatic ligament, the gastrosplenic ligament and the lesser sac . Nodal spread is extremely rare, but haematogenous spread can occur, often to the liver with nearly 50% of patients with gists presenting with metastases (fig . 2). 2gastrointestinal stromal tumour (gist): axial cect showing typical large low attenuation exophytic tumour (asterisk) arising from the greater curvature of the stomach associated with liver metastasis (arrow). Note there are no significantly enlarged regional nodes gastrointestinal stromal tumour (gist): axial cect showing typical large low attenuation exophytic tumour (asterisk) arising from the greater curvature of the stomach associated with liver metastasis (arrow). Pancreatic ductal adenocarcinoma accounts for more than 80% of malignant tumours of the pancreas, and 70% of tumours arise within the pancreatic head . Ct is the initial imaging modality used for staging of suspected pancreatic cancer with ct (along with mri and endoscopic sonography) used to distinguish between potentially resectable and non - resectable patients . For cancer at the head of the pancreas, contraindications to surgery include distant metastases, circumferential involvement of the superior mesenteric vein - portal vein segment more than 2 cm long, thrombus in the vein, and occlusion or circumferential invasion of the celiac, hepatic or superior mesenteric arteries (see table 2). Table 2summary table of local, nodal and haematogenous spread in pancreatic cancerlocal spreadtumour spreads by direct perivascular and perineural invasion;local invasion can involve the stomach, duodenum and retroperitoneum;head / uncinate process tumours: these usually extend along the sma and mesenteric root;body / tail tumours: these usually infiltrate the celiac, hepatic or splenic arterieslymph node spreadprimary drainage: superior, inferior, anterior, posterior and splenic lymph nodes;secondary drainage: porta hepatis, common hepatic, coeliac, mesenteric root lymph nodes;tertiary drainage: peri - aortic and distal superior mesenteric lymph nodeshaematogenous spreadthese usually involve the liver and peritoneal surfacesnoteearly lymphatic and haematogenous micrometastases at presentation are common summary table of local, nodal and haematogenous spread in pancreatic cancer when performing a staging ct, ideally at least two contrast - enhanced acquisitions (during the late arterial and venous phases) are advised . The pancreas is a retroperitoneal organ and has a close anatomic relationship with the abdominal peritoneal reflections . The pancreatic head is connected to the liver and lesser curvature of the stomach via the hepatoduodenal ligament and gastrohepatic ligaments, respectively . The tail of the pancreas is in continuity with the hilum of the spleen and the greater curvature of the stomach via the splenorenal and gastrosplenic ligaments . Pancreatic cancer can invade these adjacent peritoneal ligaments (as well as the transverse mesocolon) to involve the stomach, duodenum (fig . Tumour tends to spread by direct perivascular and perineural invasion within the subperitoneal space . If arising from the pancreatic head or uncinate process, tumours usually extend along the superior mesenteric artery and root of the mesentery . Biliary and pancreatic duct dilatation 3pancreatic adenocarcinoma: a axial cect showing an example of a tumour arising in the head of the pancreas (asterisk) with invasion into the duodenum . There is a focal thrombus in the portal vein (arrow); b axial cect in same patient as (a); there is associated intra- (arrows) and extrahepatic (not shown) bile duct dilatation; c axial cect showing a large bulky tumour in a different patient arising in the body of the pancreas (asterisk) encasing the origin of the celiac axis pancreatic adenocarcinoma: a axial cect showing an example of a tumour arising in the head of the pancreas (asterisk) with invasion into the duodenum . There is a focal thrombus in the portal vein (arrow); b axial cect in same patient as (a); there is associated intra- (arrows) and extrahepatic (not shown) bile duct dilatation; c axial cect showing a large bulky tumour in a different patient arising in the body of the pancreas (asterisk) encasing the origin of the celiac axis spread to regional nodes occurs, involving either peripancreatic nodes, nodes at the celiac axis and porta hepatis, or sometimes nodes further afield (table 2). Most colorectal cancers arise from adenomatous polyps, with polyps greater than 2 cm in size associated with a greater than 40% risk of malignancy . For local staging of rectal cancer, mri is now the standard imaging modality because of its increased soft tissue resolution and multiplanar capabilities . Colorectal tumours may appear as polypoid or infiltrative lesions of the bowel wall (fig . Early colonic tumours are better detected when endoluminal distension is given and the colon has been prepared, as in ct colonography . Tumour spread through the serosa appears as pericolonic fat stranding, but may be confused with an associated desmoplastic reaction (possibly mimicking diverticulitis within the sigmoid colon). The presence of fluid in the root of the sigmoid mesentery and engorgement of the adjacent sigmoid mesenteric vasculature favours diverticulitis, whilst the presence of pericolic lymph nodes is suspicious for colon cancer . Tumour spread to adjacent organs is suggested by loss of the fat planes and direct invasion (see table 3). Table 3summary table of local, nodal and haematogenous spread in colorectal cancerlocal spreadinvasion through the bowel wall into the peri - colonic fat and adjacent structureslymph node spreadfollows the vascular distribution of vessels in mesocolon;ascending mesocolon: nodes along the ileocolic vessels and right colic vessel;transverse mesocolon: nodes along the middle colic vessels;sigmoid and descending mesocolon: nodes along the inferior mesenteric vein;regional lymph nodes for rectal cancers include: mesorectal, sigmoid mesenteric, inferior mesenteric, lateral sacral, presacral, internal iliac, sacral promontory, superior rectal, middle rectal and inferior rectalhaematogenous spreadliver (via the portal vein)> lung, adrenal glands, bonesnotesin rectal cancers that have perforated the peritoneum, transcoelomic spread favours the lower right small bowel mesentery and the pouch of douglasfig . 4colonic adenocarcinoma: a axial non - contrast - enhanced ct showing tumour (asterisk) in the ascending colon with enlarged ileocolic nodes (arrows); b axial contrast - enhanced ct shows a large liver metastasis (asterisk) summary table of local, nodal and haematogenous spread in colorectal cancer colonic adenocarcinoma: a axial non - contrast - enhanced ct showing tumour (asterisk) in the ascending colon with enlarged ileocolic nodes (arrows); b axial contrast - enhanced ct shows a large liver metastasis (asterisk) nodal spread depends on the site of the primary tumour and follows the vascular distribution of the vessels within the mesocolon [11, 12]. These vessels include the ileocolic vessels and right colic vessel for the ascending mesocolon, the middle colic vessels for the transverse mesocolon, and the inferior mesenteric vein for the sigmoid and descending mesocolon . For rectal tumours, drainage is usually cranial within the mesorectum to involve the regional lymph nodes (table 3). 5) is less common, with involvement of the inguinal nodes only seen with lower rectal tumours when there is proximal lymphatic blockage (e.g. Extensive adenopathy). 5rectal adenocarcinoma: coronal reformat cect showing a bulky rectal tumour (asterisk) with enlarged right internal iliac nodes (double asterisk) and left paraaortic node (arrow) rectal adenocarcinoma: coronal reformat cect showing a bulky rectal tumour (asterisk) with enlarged right internal iliac nodes (double asterisk) and left paraaortic node (arrow) metastatic disease at presentation is uncommon (<1015% of cases). Haematogeneous spread is predominantly seen within the liver, with metastases deriving their blood supply from the hepatic artery (compared to normal liver parenchyma, which is primarily supplied by the portal vein). As a result, liver metastases imaged during the portal venous phase are seen as heterogeneous ring - enhancing metastases that are hypodense to the surrounding liver parenchyma (fig . If the primary cancer is mucinous, liver metastases may be cystic or calcified . Sites of distant metastases are determined by the venous drainage of the primary site . For example, the venous drainage of the colon and upper rectum is via the portal vein, and thus the liver is a common site of spread . However, the lower rectum has a dual drainage, with the superior haemorrhoidal vein draining into the inferior mesenteric vein and then into the portal vein, and the middle and inferior haemorrhoidal veins draining into the pelvic veins and then directly into the inferior vena cava . This explains why distal rectal cancers can result in isolated pulmonary metastases without hepatic metastases . If rectal tumours perforate the peritoneal membrane, transcoelomic spread may occur, favouring the lower right small bowel mesentery and pouch of douglas . If colorectal tumours are of mucinous histology, widespread intraperitoneal mucinous metastases may occur with characteristic scalloping of adjacent viscera (pseudomyxoma peritoneii) (fig . 6pseudomyxoma peritoneii: axial cect showing mucinous ascites exerting mass effect (scalloping) on the solid organs; primary was of gastrointestinal tract origin pseudomyxoma peritoneii: axial cect showing mucinous ascites exerting mass effect (scalloping) on the solid organs; primary was of gastrointestinal tract origin on ct, renal tumours appear as hypervascular masses, with larger lesions of heterogenous attenuation . With increasing size, they spread through the renal capsule into the perinephric fat to involve gerota s fascia . Early extension through the renal capsule is recognized by an indistinct tumour margin, thickened perirenal fascia and perinephric fat stranding . Extension into the renal vein occurs in 20% of patients at presentation with involvement of the inferior vena cava (ivc) in 510% of patients (fig . Tumour thrombus is seen as a filling defect within the vein, which should not be confused with streaming artefact from unopacified blood . The presence of venous distension on its own may be misleading as rccs are hypervascular and hence tend to have increased blood flow and venous drainage . Distinction between tumour thrombus and bland thrombus within the ivc may be difficult, although the former may have enhancing tumour vessels within it . In advanced tumours, direct invasion into adjacent structures such as the diaphragm, and posterior abdominal wall muscles may be seen (see table 4). Table 4summary table of local, nodal and haematogenous spread in renal cell carcinomalocal spreadperinephric fat; ipsilateral adrenal; adjacent viscera (including muscles);renal vein invasion (ivc)lymph node spreadvia lymphatics following the renal vessels to the ipsilateral para - aortic nodes; direct connections with the thoracic duct and mediastinum also existhaematogenous spreadcommon sites: lungs> bones, cns, adrenalsnoteextension into renal vein occurs in 20% of patients at presentation; ivc involvement in 510%fig . 7metastatic renal cell carcinoma: a coronal reformat cect showing the inferior vena cava (ivc) greatly expanded by tumour thrombus (arrows) with associated enlarged mediastinal nodes (asterisks). The primary renal tumour has been excised; b axial cect showing enhancing lesion in the left gluteus maximus muscle in the same patient, which increased in size on interval ct studies, in keeping with a metastatic deposit; note the numerous venous collaterals in the anterior abdomen (short arrows) due to the ivc thrombus; c axial cect shows a large lytic deposit in the lumbar spine (asterisk) in the same patient causing compromise of the spinal canal and cord (arrows) summary table of local, nodal and haematogenous spread in renal cell carcinoma metastatic renal cell carcinoma: a coronal reformat cect showing the inferior vena cava (ivc) greatly expanded by tumour thrombus (arrows) with associated enlarged mediastinal nodes (asterisks). The primary renal tumour has been excised; b axial cect showing enhancing lesion in the left gluteus maximus muscle in the same patient, which increased in size on interval ct studies, in keeping with a metastatic deposit; note the numerous venous collaterals in the anterior abdomen (short arrows) due to the ivc thrombus; c axial cect shows a large lytic deposit in the lumbar spine (asterisk) in the same patient causing compromise of the spinal canal and cord (arrows) lymphatic spread from rcc tends to follow the renal veins to involve the ipsilateral para - aortic nodes . There are also direct connections with the thoracic duct and mediastinum, which can account for the rare presence of mediastinal and hilar node involvement (fig . Metastatic involvement in renal cell carcinoma is seen (in order of decreasing frequency) in: the lung (5060%), bone (3040%), liver (3040%), adrenal gland (5%), contralateral kidney (5%), retoperitoneum (5%) and brain (5%). More unusual sites that have been reported include the pancreas, peritoneum, bowel, thyroid and muscle (fig . Cross - sectional imaging (ct or mri) is used for preoperative tumour assessment, with ct being the imaging modality of choice for assessing metastatic disease . Bowel opacification is usually achieved with a positive oral contrast medium (e.g. Dilute gastrograffin), which is essential for the detection of peritoneal deposits . Intravenous contrast is also routinely administered unless contraindicated, with imaging performed in the portal venous phase . Conversely, local pelvic spread is better assessed with pelvic mri than ct (see table 5). Table 5summary table of local, nodal and haematogenous spread in ovarian carcinomalocal spreaduterus and broad ligament (via the fallopian tube);direct invasion of the rectum, colon, bladder and pelvic side walllymph node spreadvia lymphatics travelling along with the ovarian vessels to terminate in common iliac and para - aortic nodes;via the broad ligament to terminate in the internal iliac and obturator nodes;via the round ligament to terminate in the external iliac and inguinal nodestrans - coelomic spreadcommon sites: undersurface of the diaphragm, liver surface, pouch of douglas, omentum, serosal bowel surfaceshaematogenous spreadthis occurs late during the disease;liver> lungs, kidney, bonenoteascites arise from increased production from tumour surfaces and/or occlusion of retroperitoneal lymph nodes summary table of local, nodal and haematogenous spread in ovarian carcinoma primary ovarian cancer has variable appearances, presenting either as a solid mass or mixed solid / cystic lesion (fig . Ovarian cancer spreads locally to adnexal structures, the uterus and the contralateral ovary, although bilateral ovarian cancer may occur in 1150% of cases . Invasion of the pelvic side wall is indicated when the tumour is seen encasing the iliac vessels or is within 3 mm of the pelvic sidewall . 8ovarian carcinoma: coronal reformat cect showing a large solid / cystic adnexal mass arising from the pelvis (asterisk), with large - volume para - aortic adenopathy (double asterisk), ascites and small peritoneal deposits (arrows). Although para - aortic nodal involvement is seen in metastatic ovarian carcinoma, the large volume is rather unusual; b sagittal reformat cect showing subcapsular deposits over the liver in a different patient (arrows); c axial cect in the same patient as (b) showing a thick omental cake (arrows); d axial cect in the same patient as (b) showing multiple enlarged (short axis diameter> 5 mm) cardiophrenic nodes (arrows) ovarian carcinoma: coronal reformat cect showing a large solid / cystic adnexal mass arising from the pelvis (asterisk), with large - volume para - aortic adenopathy (double asterisk), ascites and small peritoneal deposits (arrows). Although para - aortic nodal involvement is seen in metastatic ovarian carcinoma, the large volume is rather unusual; b sagittal reformat cect showing subcapsular deposits over the liver in a different patient (arrows); c axial cect in the same patient as (b) showing a thick omental cake (arrows); d axial cect in the same patient as (b) showing multiple enlarged (short axis diameter> 5 mm) cardiophrenic nodes (arrows) peritoneal dissemination is the commonest mode of spread, being found in approximately 70% of patients at presentation . Common sites include the greater omentum, paracolic gutters, the pouch of douglas, the liver capsule (fig . Less commonly, implants may be seen within the mesentery, along the porta hepatis, lesser sac, splenic surface and gastrosplenic ligament [1618]. Ascites are often present, due to either increased production from tumour surfaces or reduced peritoneal resorption from invasion of lymphatics by tumour cells . Peritoneal deposits on ct have a variety of appearances including enhancing nodular soft tissue lesions, linear / plaque - like thickening of peritoneal reflections (fig . 8c), tiny calcifications or mixed solid and cystic or purely cystic lesions [19, 20]. The involvement of bowel serosa can lead to (asymmetric) bowel wall thickening, tethering of bowel loops and bowel obstruction . Liver surface implants (subcapsular deposits) may be seen, with deposits causing characteristic scalloping of the liver capsule . Metastatic mucinous tumours of the ovary can lead to pseudomyxoma peritoneii, although in many of these cases, the primary mucinous tumour actually arises from the gastrointestinal tract with secondary involvement of the ovary . Lymphatic spread occurs via three routes and is usually seen in conjunction with peritoneal spread . Routes include: (1) along the ovarian vessels to the common iliac and para - aortic nodes (fig . 8a); (2) via the broad ligament and parametria to the internal iliac and obturator nodes; and (3) rarely via the round ligaments to the external iliac and inguinal nodes . Cardiophrenic nodes (defined as> 5 mm short axis diameter on ct) are characteristically involved as they are the main lymphatic drainage route from the peritoneal cavity (fig . Haematogenous dissemination is rare in ovarian carcinoma with a malignant pleural effusion being the most common manifestation . At presentation, other sides of spread include the spleen, kidneys, adrenals, lungs, brain and bone . Mri is traditionally used for local staging of prostate cancer because of its high contrast resolution (as in rectal cancer). It is able to differentiate organ - confined disease from tumour that demonstrates extracapsular spread . Direct involvement of the seminal vesicles or adjacent organs is also well seen on mri . Ct is used for nodal and distant staging of prostate cancer (see table 6). Table 6summary table of local, nodal and haematogenous spread in prostate cancerlocal spreaddirect extension through the prostate capsule into the seminal vesicles and bladder baselymph node spreadorder of nodal involvement: obturator, presacral, internal iliac, common iliachaematogenous spreadbone>> lung and liver;spinal bone metastases are the commonest site (due to the direct communication between the presacral and periprostatic veins)notesdenonvillier s fascia forms a relative natural barrier to rectal spread;tumours at the apex of the prostate are more likely to demonstrate extracapsular extension because of relatively little capsule at this level summary table of local, nodal and haematogenous spread in prostate cancer classically, two lymph node metastatic patterns have been reported . 9a) and para - aortic lymph nodes, and this pelvic nodal involvement is sequential with spread to the obturator nodes, presacral nodes, internal iliac and common iliac nodes . Pattern 2 involves para - aortic nodes only, with this second pattern of nodal spread felt to be due to haematogenous dissemination . 9prostate carcinoma: a coronal reformat cect showing irregular enlarged prostate tumour (white arrow) extending into the bladder with enlarged left pelvic side wall node (black arrow), liver metastases (double arrows) and lesion in the left pubic ramus with large soft tissue component (*). Note the bones are sclerotic in keeping with diffuse bony metastases; b sagittal reformat cect in a different patient on bone windows, showing multiple sclerotic metastases in the thoracolumbar spine prostate carcinoma: a coronal reformat cect showing irregular enlarged prostate tumour (white arrow) extending into the bladder with enlarged left pelvic side wall node (black arrow), liver metastases (double arrows) and lesion in the left pubic ramus with large soft tissue component (*). Note the bones are sclerotic in keeping with diffuse bony metastases; b sagittal reformat cect in a different patient on bone windows, showing multiple sclerotic metastases in the thoracolumbar spine distant spread occurs in one third of patients with involved sites including bone (90%), lung (50%), liver (25%) (fig . Osteoblastic sclerotic metastases rather than lytic bone lesions are usually seen on ct . In order of decreasing frequency, skeletal metastases are seen within the vertebrae (fig . Spinal involvement is initially more common within the lumbar region and less likely within the cervical spine (97% cf . Pulmonary metastases occur in between 527% of patients at presentation and are more often seen in association with the second pattern of nodal involvement . Pulmonary involvement can either appear as lymphangitis carcinomatosa (due to direct invasion of the pulmonary lymphatics) or as pulmonary nodules (due to haematogenous spread) [22, 24]. This review has described the typical patterns of tumour spread for the more common cancers occurring within the abdomen and pelvis . Knowledge of the characteristic sites of spread in such cancers is felt to be essential for the radiologist when reporting staging and restaging ct scans . It is hoped that the summary tables in particular will act as a useful aide - mmoir to the reporting radiologist . Correct identification of metastatic sites of disease is important, as this will not only have an impact on patient management, but also a bearing on patient prognosis.

The long - term use of immunosuppressive agents for prevention of allograft rejection increases the risk of malignancy approximately 100 times as high as that in the general population . The prevalence rate of post - transplant malignancies in total differs between geographical areas; for example, in europe, that rate is 1.6% and in australia is 24%, with a mean of 6% . Skin cancers, mostly squamous cell carcinoma (scc), are the most common tumors among persons have solid organ transplantation . But, however, iranian studies found that the most common malignancy after kidney transplantation was kaposi sarcoma (ks) among the iranian patients . Ks is a skin tumor of multicentre origin, characterized histologically by endothelium - lined vascular spaces and spindle - shaped cells . Ks presents as single or multiple lesions on mucosal surfaces, including the skin, lungs, gastrointestinal tract and lymphoid tissues . The etiopathogenes of ks is complex and poorly understood, but is almost certainly dependent on human herpes virus type 8 (hhv -8) infection in immunosuppressed, immunogenetically susceptible individuals . Although the treatment of ks is controversial, it should ideally address these pathogenic issues . The current guideline is reduction of immunosuppression as first - line treatment, but these recommendations are based on anecdotal experience or uncontrolled studies . Perhaps the most fundamental controversy that has implications for all aspects of the disease surrounds the nature of ks: i.e. Whet her it is a true malignancy or reversible hyperplasia . The aim of present study was to investigate the frequency of ks in patients with kidney transplantation in 21 years period . An observational prospective follow - up study with a retrospective component, carried out in the imam reza hospital from kermanshah university of medical sciences (kums) during the period 19912012 . Patients with pre - transplant neoplasm will be excluded from the analysis (n = 46). Patients who had received transplants were identified through the hospital's transplant registry . For each patient, information includes donor and recipient characteristics, patient and graft survival and cancer incidence after transplantation, data of serologic tests such as hiv were received . The period of follow - up for each patient starts on the day of transplantation and continues until death or last reported contact . Following the methodology used in similar studies, patients will not be removed from the analysis at the time of graft failure for several reasons . We usually suspect ks when a kidney recipient presents with multiple hyperpigmented cutaneous nodules that may be associated with gastrointestinal discomfort and pulmonary symptoms resistant to conventional therapies . Then a battery of endoscopic, bronchoscopic, radiologic and pathologic tests is used to diagnose ks . There were two main distinct periods of immunosuppressive regimen: the first period was from 1991- 2002 azathioprine, cyclosporine and prednisolone . The second period was from 2002 onwards during which the patients received triple immunosuppressive therapy consisting of cellcept, cyclosporine and prednisolone at the same dosages mentioned above . Induction therapy using anti - thymocyte globulin (atg) or anti - lymphocyte globulin (alg) was preserved to the high risk patients in the early phase of transplantation or treatment of acute rejection; okt-3 was not used in any of the studied populations . The doses of immunosuppressive agents were reduced, with changed drugs to sirolimus, or the agents were withdrawn upon diagnosis of ks . The method of reduction of immunosuppression and decision on which the agent to be reduced or withdrawn were dependent on the individual patient's health condition, response to treatment and his / her physician's judgment . From march 1991 to december 2012, 1487 kidney transplantations were performed . Among them, 67 malignant diseases were diagnosed in 64 patients with an overall incidence of 4.5% and ks was diagnosed as more frequent malignancy in 17 (25.37%) patients (table 1). Of these 17 ks patients, 10 were males and 7 were female with median age of 47.8 years old . The mean time between transplantation and non - ks malignant tumors was 34.4 21.8 months (range 12140 months), while in ks patients it was 18.7 25.2 months, which was statistically significantly different (p <0.05). After detection of ks in 12 patients, we perform serum antibody detection against hhv . Among them, 8 (66.6%) were seropositive . Data of characteristics of transplant patients with kaposi's sarcoma were summarized in the table 2 . Type and frequency of most frequent post - transplant malignancies (n = 67) characteristics of transplant patients with kaposi's sarcoma there was no significant increase in the number of ks patients under cellcept compared to azathioprine or in the interval to development of ks . The sites of cutaneous involvement were lower extremities in 9 (52.9%), followed by upper - limb involvement in 5 (29.41%) patients . Other ks lesions occurred on trunk in 4 (23.5%) and hard palate in 2 (11.7%) patients . Immunosuppression was reduced in 10 (58.8%) patients (that 8 (80%) patients of them received sirolimus) and thoroughly withdrawn in the remainder (including three cases of visceral involvement). In 2 (11.7%) patients immunosuppression was discontinued . In 11 (91 .6%) of 12 patients, ks skin lesions improved with therapy (excluded are five patients who died soon after ks diagnosis). Patients received additional therapy arbitrarily if, in the opinion of the treating clinician, lesions were not improving . In the patients with hhv seropositive, ganciclovir were prescribed . The renal prognosis of patients who did not succumb to disseminated disease was related to the management of immunosuppression . All two patients in whom immunosuppression was discontinued had functioning grafts when ks was diagnosed, but all grafts were acutely rejected . One patient had azathioprine and one patient had cellcept immunosuppressive therapy and there is no significant difference between these two regimens (p> 0.05). Dialysis was re - instituted in these patients a mean of 5 weeks after discontinuation of all immunosuppression . Other patients had good response to reducing immunosuppression and remained their grafts in period of study . From march 1991 to december 2012, 1487 kidney transplantations were performed . Among them, 67 malignant diseases were diagnosed in 64 patients with an overall incidence of 4.5% and ks was diagnosed as more frequent malignancy in 17 (25.37%) patients (table 1). Of these 17 ks patients, 10 were males and 7 were female with median age of 47.8 years old . The mean time between transplantation and non - ks malignant tumors was 34.4 21.8 months (range 12140 months), while in ks patients it was 18.7 25.2 months, which was statistically significantly different (p <0.05). After detection of ks in 12 patients, we perform serum antibody detection against hhv . Among them, 8 (66.6%) were seropositive . Data of characteristics of transplant patients with kaposi's sarcoma were summarized in the table 2 . Type and frequency of most frequent post - transplant malignancies (n = 67) characteristics of transplant patients with kaposi's sarcoma there was no significant increase in the number of ks patients under cellcept compared to azathioprine or in the interval to development of ks . The sites of cutaneous involvement were lower extremities in 9 (52.9%), followed by upper - limb involvement in 5 (29.41%) patients . Other ks lesions occurred on trunk in 4 (23.5%) and hard palate in 2 (11.7%) patients . Immunosuppression was reduced in 10 (58.8%) patients (that 8 (80%) patients of them received sirolimus) and thoroughly withdrawn in the remainder (including three cases of visceral involvement). In 2 (11.7%) patients immunosuppression was discontinued . In 11 (91 .6%) of 12 patients, ks skin lesions improved with therapy (excluded are five patients who died soon after ks diagnosis). Patients received additional therapy arbitrarily if, in the opinion of the treating clinician, lesions were not improving . In the patients with hhv seropositive, ganciclovir were prescribed . The renal prognosis of patients who did not succumb to disseminated disease was related to the management of immunosuppression . All two patients in whom immunosuppression was discontinued had functioning grafts when ks was diagnosed, but all grafts were acutely rejected . One patient had azathioprine and one patient had cellcept immunosuppressive therapy and there is no significant difference between these two regimens (p> 0.05). Dialysis was re - instituted in these patients a mean of 5 weeks after discontinuation of all immunosuppression . Other patients had good response to reducing immunosuppression and remained their grafts in period of study . Post - transplantation ks is a well - known complication after renal transplantation with a possible negative impact on the patient's and graft long term survival . The ks incidence peaks during the first year post - transplantation . In our study, 82.3% of all ks cases were diagnosed in the first 2 years after receiving a renal allograft, which is compatible with previous studies . The results of this study showed lower incidence of ks (1.1%) in our transplant population than that reported from other regional countries . Further, ks is frequent in african renal transplant recipients, where 13.3% of all transplanted patients developed ks . We investigated the ks was the most frequent post - transplantation malignancies . In confirm to our study, saudi arabian and turkish studies were reported the most frequent post - transplantation malignancies were ks with 87.5% and 80.0%, respectively . Also, nafar et al ., found that the most common malignancy after kidney transplantation was ks among the iranian patients . The risk of death from dissemination of malignancy should be weighed against the risk of graft rejection . According our results, in 11 patients (91.6%) ks skin lesions improved with reduction (including 6 patients with sirolimus therapy) or discontinued of immunosuppression with a graft loss rate of 11.7% . In compare to our study, reduction of immunosuppression resulted in complete remission of ks in 28% of patients from saudi arabia, and 61% of italian patients . Duman et al ., report on complete remission after reduction of immunosuppressive drugs in all patients [12/12], with a graft loss rate of 20% in turkey . Reduction of immunosuppression allows for the immune system to reduce viral replication producing clinical remission of disease . New antiviral agents have recently been introduced as a promising therapeutic option in patients with ks . However, prospective studies that will determine the efficacy of this approach are warranted . Sirolimus is a potent immunosuppressive drug that has been recently reported as an effective agent in the treatment of ks . Cutaneous ks lesions disappeared in all patients three months from the initiation of sirolimus therapy . Sirolimus may become the first choice immunosuppressant in renal transplant recipients with ks for pro - viding optimal immunosuppression and inhibiting the progression of malignancy . Hhv-8 has been described in patients with hiv infection and ks . The results of serologic studies support the notion that infection with hhv-8 is nearly universal in patients with ks, since specific antibodies are detectable in 70% to 90% of all patients with ks and almost 100% of immunocompetent patients with the disease . Also it's clear that pre - transplantation hhv-8 seropositivity is found to be associated with an increased risk of post - transplant ks . Immunosuppressive treatment may induce reactivation of latent virus infection, playing an important role in the development of combined iatrogenic and endemic ks . It is also possible that hhv-8 may be transmitted from the donor to induce sarcoma development in the organ recipient . We investigated that about 67% of our ks patients were seropositive for hhv after transplantation . Laboratory studies of the susceptibility of hhv-8 to antiviral drugs suggest that the virus is resistant to acyclovir and penciclovir, but sensitive to ganciclovir, foscarnet and cidofovir . Ks is a common long - term complication in renal transplant recipients, with an increased incidence compared with the general population . Given that candidates for organ transplantation who are seropositive for hhv-8 -and thus at risk for ks- can now be identified, chemoprevention should be available in this high - risk population . Such strategies in hhv-8-seropositive candidates for organ transplantation should be directed against the virus itself, and the immunosuppressive regimen should be carefully monitored to avoid the possibility of rejection.

Breast cancer is the most common cancer among american women, except for skin cancers . The chance of developing invasive breast cancer at some time in a woman's life is a little less than 12% . It is the second leading cause of cancer death in women, exceeded only by lung cancer . The chance that breast cancer will be responsible for a woman's death is about 3% . Although clinical signs of disseminated disease occur in fewer than 10% of women at the time of diagnosis, the disease relapses in the form of metastasis within 5 years of surgery in about half of apparently localized tumors . It is difficult to predict the occurrence of distant metastases since breast cancer is a heterogeneous disease encompassing complex pathologic entities . A dynamic interaction between tumors and the immune system is essential for tumor survival, growth, and metastasis . Tumors are infiltrated with large number of immune cells that constitute a major cell population in the tumor microenvironment . Tumor cells depend on their microenvironment to provide signals for growth, anti - apoptosis, angiogenesis, and metastasis . However, tumor cells are also under the surveillance due to their recognition by immune cells as foreign . Analysis of the interactions between tumor cells and the host's immune system has led to the realization that tumor cells have devised multiple strategies to evade immune attack . Development of an invasive cancer, however, is not only a result of the genetic changes in the tumor cell but also the result of genetic and epigenetic changes within the host . Host cells, including inflammatory cells, endothelial cells, and fibroblasts, are recruited and activated in the microenvironment of transformed cells . The acute inflammatory response might succeed in eliminating the malignant cells, but if not, a chronic inflammatory process develops in conjunction with the dying tumor cells . The subsequent reciprocal interactions between these responding normal host cells and genetically altered cells result in the development of an invasive cancer . There is a constant interplay between the innate and adaptive immune systems, which leads to a protective immune response against pathogens and transformed cells and contributes effectively to discrimination between self and nonself . Persistent protumor immune responses (inflammation), now generally accepted as initiating primary tumor development, are also being recognized as mediators of cancer metastasis . Thus, novel anticancer therapeutic strategies targeting molecular and/or cellular mechanisms regulating these collaborative interactions may provide efficacious relief for metastatic disease . This paradox was first resolved by matzinger in 1994, who proposed that our immune system is designed to combat danger, rather than mediate recognition of nonself over self . Pathogen - associated molecular patterns (pamps) and endogenous molecules created upon tissue injury, since called damage - associated molecular patterns (damps), signal the threat of either infection or injury to the organism, independently of their nonself- or self - identity [710]. Damage - associated molecular patterns (damps) include endogenous intracellular molecules released by activated or necrotic cells and extracellular matrix (ecm) molecules that are upregulated upon injury or degraded following tissue damage . Among the cellular receptors that sense these danger signals, toll - like receptors (tlrs) represent a key molecular link between tissue injury, infection, and inflammation . Tlrs are critical in bridging innate and adaptive immune responses and play a significant role in cancer immunosurveillance . Innate immune cells including natural killer (nk), natural killer t (nkt), and t cells play a critical role in protecting the host against cancer . Macrophages and dendritic cells (dcs), in particular, function as major sensors of invading pathogens and transformed cells via the tlrs . Adaptive immunity is crucial to the elimination of pathogens and tumor cells in the late phase of host defense responses and generates more specific tumor immunity and immunological memory . Tlrs are known to regulate cancer immunity and tolerance by controlling the suppressive function of regulatory t (treg) cell and through innate immune responses mediated by other immune cells [1113]. Tlr signaling, critical for innate and adaptive immune responses, has been thought to be restricted to immune cells . However, many studies suggest that tumor cells bear tlrs and that tlr signaling promotes tumor growth and immune evasion [1517]. Tlr activation by damps may initiate positive feedback loops where increasing tissue damage enhances proinflammatory responses leading to chronic inflammation . As tlrs are widely expressed on tumor cells and immune cells and play important roles in the initiation and progression of cancer, they may thus serve as an important target and have an effective perspective on breast cancer treatment . Tlrs 1, 2, 4, 5, and 6 are expressed on the cell surface; tlrs 3, 7, 8, and 9 are found almost exclusively within endosomes . Different tlrs exhibit specificity for pathogen - derived ligands; tlrs 2, 3, 4, 5, 7, and 9 recognize bacterial lipoproteins, double - stranded rna / poly (i: c), lipopolysaccharides (lps), flagellin, single - stranded rna, and cpg - containing dna, respectively [1823]. Tlr10 is expressed in humans but not in mice, tlr8 is not functional in mice and tlrs 11, 12, and 13 are expressed in mice but not in humans . There are several studies which suggest that damp - mediated inflammation plays a vital role . Necrotic cells were found to induce proinflammatory and tissue repair gene synthesis and cause dc maturation in a tlr2-dependent manner, as a result of the release of their intracellular contents . Other intracellular molecules such as heat shock proteins including hsp70, gp96, hsp22, and hsp72 and high - mobility group box-1 protein (hmgb1) as well as ecm molecules such as biglycan, tenascin - c, versican, and fragments of ecm molecules including oligosaccharides of hyaluronic acid (ha) and heparan sulfate (hs) have been shown to activate tlrs . Tlr1, along with tlr2, was found to be important for the activation of professional antigen - presenting cells by -defensin-3, a host - derived antimicrobial peptide . Self - nucleic acids have also been described as endogenous danger signals, namely, mrna recognized by tlr3, single - stranded rna (ssrna) sensed by tlr7 and 8, and igg - chromatin complexes recognized by tlr9 . Tlr2, 4, 7, and 8 were shown to be activated by antiphospholipid antibodies (apl) isolated from patients with apl syndrome . The signaling pathways utilized by various tlrs differ, which results in varied cellular responses . For example, tlr3, the receptor for double - stranded rna couples to the adaptor protein trif . In contrast, other tlrs couple to the adapter myeloid differentiation primary response gene 88 (myd88) [25, 26]. The traf6 in turn activates tak1 that phosphorylates and activates the ikk complex resulting in the release and translocation of nf-b to the nucleus . Tak1 also activates stress - activated protein kinase (sapk) pathways and activates c - jun - nh2-kinases (jnk) and p38 . The myd88-coupled tlrs induce the synthesis of cytokines such as tnf-, il-6, and il-1, key mediators of the inflammatory response [27, 28]. Tlr4, the receptor for lps, is unique in that it activates both myd88-dependent and trif - dependent pathways . The link between inflammation and cancer is well documented [29, 30]. Several inflammatory diseases, including inflammatory bowel disease, increase the risk of cancer . Conversely, in tumors that are epidemiologically unrelated to overt inflammatory conditions (such as breast cancer), the activation of oncogenes can trigger the production of inflammatory molecules and the recruitment of inflammatory cells . In the tumor microenvironment, inflammatory cells and molecules influence almost every aspect of cancer progress, including the metastatic ability of tumor cells . There is biological heterogeneity among tumors with regard to cellular infiltrates, identifying subsets of mononuclear inflammatory cells both at the tumor centre and at the invasive front, which seem to be associated with the occurrence of distant metastasis . Intratumour leucocytes from peripheral blood penetrate the tumor architecture after their phenotypic modification, from the invasive front to the tumor centre . This seems to be a dynamic process in which inflammatory cells and immunomodulatory mediators present in the tumor microenvironment polarize the host immune response towards specific phenotypes impacting on tumor progression . Previously, there were six recognized hallmarks of cancer, namely, unlimited replicative potential, self - sufficiency in growth signals, insensitivity to growth inhibitors, evasion of programmed cell death, ability to develop blood vessels, and tissue invasion and metastasis . A group of cytokine proteins, including il-1, il-6, tnf-, and rankl, activate inflammation and are known to augment tumor cells' ability to metastasize by affecting several steps in the cells' dissemination and implantation at secondary sites [29, 32, 33]. Inflammatory cytokines lie downstream of the master gene transcription factor nf-b, for promoting inflammation which is itself activated by them . There is strong evidence that the tumor microenvironment is inflammatory and that activation of the innate immune system plays a role in the progression of cancer [34, 35]. A major source of inflammatory cytokines in the tumor microenvironment, are specialized white blood cells called macrophages . Tumor - associated macrophages assist the malignant behaviour of tumor cells, not only by producing cytokines, but also by secreting growth factors and matrix - degrading enzymes [3638]. It has long been suggested that there may be common pathways of inflammation shared by responses to infection and to malignancy . Recent evidence indicates that tlrs on macrophages may be critical elements in these common pathways . Myd88 has been reported to activate not only ap-1 and nf-b subunit p65 and p50, but also c - rel, c / ebp, and c / ebp. In case of lps signaling through tlr4, where nf-b and ap-1 activities are relatively preserved in myd88-deficient macrophages, the specific defect in c - rel and the profound defect in c / ebp/ activation likely accounts for the reduction of il-12 p40, il-6, and tnf. The absence of both c / ebp/ specifically in tlr signaling impairs key proinflammatory cytokines without affecting other nf-b - dependent genes such as ib . Toll - like receptors are expressed on cells of the immune system but there is growing evidence that tlrs are also expressed on tumor cells, where they may influence tumor growth and host immune responses . Activation of tlrs expressed on tumor cells may have profound consequences for tumor growth by factors released after tlr activation . Tumor immune evasion may be facilitated by inhibitory cytokines, inflammatory factors, proteinases, and other small molecules such as nitric oxide . Recent evidence suggests that tlrs also contribute to tumor - cell resistance to apoptosis and increased invasiveness . The human breast cancer cell line mda - mb-231 was found to express tlr1-tlr10 at both the mrna and protein levels . Knockdown of tlr4 gene in mda - mb-231 resulted in a dramatic reduction of breast cancer cell viability and inhibition of il-6 and il-8 cytokines compared with vector control . Another study highlights the role of tlr9 in highly invasive mda - mb-231 breast cancer cell line which when activated promotes mda - mb-231 cell invasion by increasing the activity of matrix metalloproteinase 13 (mmp13), but not mmp8 . Samples of mammary carcinomas with recurrence have also exhibited a significant increase in the mrna levels of tlr3, tlr4, and tlr9 . A significant percentage of tumors also showed tlr4 expression by mononuclear inflammatory cells (21.6%) and tlr9 expression by fibroblast - like cells (57.5%). Tumors with high tlr3 expression by tumor cell or with high tlr4 expression by mononuclear inflammatory cells (mics), but not tlr9 high fibroblast like cells were significantly associated with higher probability of metastasis . This study highlights the importance of the tumor stromal cells in tumor behavior, and how tlr - induced inflammation on inflammatory cells drives metastatic cascade . Synthetic tlr9-ligands (cpg - sequence containing oligonucleotides) stimulated tlr9 expressed on cancer cells as well as various normal cells, including mesenchymal stem cells and stimulated their invasion in vitro . This invasion was mediated via downregulation of tissue inhibitor of matrix metalloproteinase-3 (timp-3) and through matrix metalloproteinase-13 (mmp-13) activation . Expression of tlr9 isoforms a and b have been detected in clinical breast cancer specimens . Expression of tlr9 and its invasive effects on breast cancer cells has been found to be regulated by estrogen receptor- (er) and sex steroid hormones . Tlr9 expression was also found to be affected by commonly used hormonal cancer therapy bicalutamide . Activation of tlr signaling on tumor cells by their ligands can also trigger apoptosis and may have therapeutic effects . For example, in a randomized clinical trial for the efficacy of poly (a: u) dsrna, therapeutic effect was mediated through tlr3 expressed on tumor cells, and could therefore represent an effective targeted treatment in patients with tlr3-positive cancers . The predictive value of tlr3 expression by tumor cells for the efficacy of poly (a: u) dsrna was determined in 194 breast cancer patients enrolled in a randomized clinical trial . However, conventional chemotherapy or in vivo injection of poly (a: u), alone or in combination, failed to reduce tumor growth unless an immune - chemotherapeutic regimen of vaccination against tumor antigens was included . Recently, tlr5 has been found to be highly expressed in breast carcinomas and activation of tlr5-signaling pathway was found to be overly responsive in breast cancer cells by inhibiting cell proliferation and an anchorage - independent growth . In addition, the secretion of soluble factors induced by flagellin, was found to the growth - inhibition of breast cancer cells in an autocrine fashion . This inhibitory activity was further confirmed in vivo using mouse xenografts models of human breast cancer cells . Sites of chronic inflammation are often associated with the establishment and growth of various malignancies including breast cancer . Enhanced neutrophilic and granulocytic infiltration in lungs and bone of the proarthritic and arthritic mice and subsequent increase in circulating levels of proinflammatory cytokines, such as macrophage colony stimulating factor (m - csf), interleukin-17 (il-17), interleukin-6 (il-6), vascular endothelial growth factor (vegf), and tumor necrosis factor - alpha (tnf - alpha) were found to contribute to the increased metastasis . Breast cancer - associated secondary metastasis was found to be significantly increased in pro - arthritic and arthritic conditions . Breast cancer metastasis was found to be significantly reduced by blocking the il-17 and cox-2 pathway . Inflammatory tlr signaling has also been shown to promote the attraction and generation of th17 cells induced by tumor cells and tumor - derived fibroblasts . Enhanced migration of th17 cells to tumor sites was reported to be due to the expression of chemokines and tumor - derived fibroblasts . There are currently approximately twenty drugs in preclinical development, with a further dozen or so in clinical trials . There are clearly many options for the targeting of tlrs, because the key function of tlrs is to induce cytokines, which are well validated in these diseases and are successfully being targeted in the clinic . Tlrs occur early in pathways and so inhibiting them might be more potent than inhibiting their downstream cytokine targets . Are a feasible option, but only for those on the cell surface, such as tlr2, tlr4, and tlr5 . Small - molecule antagonists (e.g., eritoran against tlr4 or odn - based inhibitors of tlr7) might be a better prospect, but it is hard to predict their off - target effects and efficacy . Because there are kinases on the signaling pathways, these might also be sensitive to inhibition . One major concern here, however, is that such inhibitors might block multiple tlrs and therefore give rise to unwanted immunosuppression . Studies on knockout mouse indicate that there is less redundancy in tlrs in relation to inflammation . Imiquimod is already approved for its antiviral effects, whereas mpl is approved as a vaccine adjuvant . In terms of antagonism, effects of tlr inhibitor, eritoran have been found to be significant but somewhat marginal . To further develop more effective tlr therapeutic targeting strategy, there are a few more tasks: further identifying and determining the pathogenesis of challenging medical conditions like cancer; analysis of genetic sequence, molecular structure, epigenetic observations, and functional activities on both animal model and human clinical studies; design of clinical study based on study indication, dosing regimens, drug delivery route or format consideration, and pharmacokinetics; timely and objective assessment of adverse events with details . Targeting tlrs will therefore in all likelihood prevent the induction of many immune and inflammatory proteins . The wide tissue distribution of tlrs, however, may make it difficult to determine whether an agonist or an antagonist will be most effective therapeutically . Metastasis is regulated not only by intrinsic genetic changes in malignant cells, but also by the microenvironment . Several studies have demonstrated that sites of chronic inflammation are often associated with the establishment and growth of various malignancies . Toll - like receptors (tlrs) have emerged as sensors that can detect a variety of invading pathogens and malignant cells . Since their discovery a decade ago, tlrs have been shown to be critical for efficient innate and adaptive immunity and the framework of tlr - mediated signaling pathway has been explained . However, tlr activation may be a two - edged sword, with both antitumor and pro - tumor consequences . The general expression of functionally active tlrs by tumor cells and inflammatory cells in the stroma by putative endogenous ligands suggests that tlr signaling may be continually activated and may contribute to tumor progression and metastasis . Understanding tlr function in tumor biology may lead to discovery of new therapeutic targets in cancer therapy.

Forensic odontology has established itself as an important and often indispensible science in medicolegal matters and identification of the dead . The forensic importance of dental tissue has been well recognized because tooth is the hardest of all human tissues . They are well preserved for a long period even after death, hence dental remains are the most stable biological evidence encountered in crime cases and yield useful information . Blood grouping has been one of the major factors for identification of biological materials in forensic investigations and is a widely used technique in forensic laboratories . The presence of abo blood group and rhesus factor is applied to inherited antigens detected on red cell surface by specific antibodies . Once the blood group and rhesus factor are established, it remains unchanged throughout life . Kind in 1960 discovered the presence of abo blood group in saliva by absorption - elution (ae) method . Blood grouping from dried stain by elution procedure was described more than 50 years ago but not employed widely in forensic serology, until 1960, when kind refined this technique . Ae technique / procedure originally devised by siracusa is now employed in all forensic laboratories because it is proven to be most sensitive, reliable and reproducible . Pulp tissue is one of the most protected tissues being surrounded from all sides by dental hard tissues . Pulp contains numerous blood vessels and blood group antigens are certainly present in tooth pulp . The distribution of abo substances from the pulp cavity wall to the dentin edge and the enamel gradually reduces, because of fewer possibilities of diffusion of antigens from both blood and saliva . The existence of blood group antigens in tooth dentin and enamel and their nature has been substantiated by infusion sedimentation phenomena combined with inherently present antigens . This theory describes the infusion of water - soluble antigens from saliva into the tooth tissue . The presence of abo blood group and rhesus factor antigen in soft and hard dental tissues makes a possible contribution in human identification even in decomposed bodies . Therefore, blood group and rhesus factor determination for biological evidence on tooth material is of great importance in forensic odontology . The aim of this study was to determine the abo blood grouping and rhesus factor from dentin and pulp of extracted teeth using ae technique at 0, 3, 6, 9 and 12 months after extraction . A brief case history with relevant medical history was recorded from patients selected for study and consent was taken . The blood groups were determined for all the study participants using capillary blood by slide agglutination method . Carious teeth and grossly decayed teeth were excluded; and teeth extracted for periodontal, and orthodontic purposes were included in the study . The extraction procedure was carried out under local anesthesia following the aseptic protocol in the department of oral and maxillofacial surgery . The extracted teeth were dried and stored in labeled bottles for a span of 3, 6, 9 and 12 months . The pulp was scooped with a spoon excavator and dentin was powdered using a straight fissure bur [figure 1]. The blood grouping from teeth was performed by ae technique using powdered dentin and dental pulp . The pulverized dentin powder and pulp were divided into three equal parts and were taken into six sterile test tubes containing 2 ml of saline and labeled, respectively . To each of these test tubes, three drops of antiserum a, b, d was added respectively, and the test samples were sufficiently soaked with antiserum for 2 h and left standing at room temperature . Each sample was washed five times with cold saline solution by centrifuging it at 3000 rpm for 5 min and the supernatant was removed with pipette . Then two drops of fresh saline were added to the sample, and the test tubes were heated in a water bath at a temperature of 5055c for 10 min to elude the antibodies . (a) dissected teeth samples, (b) pulverized dentine and (c) dried pulp tissue a drop of 0.5% red cell suspension of known blood group a, b and o was freshly prepared and immediately put into respective test tubes . The samples were incubated at 37c for 30 min to enhance agglutination, and then, it was centrifuged at 15002000 rpm for 1 min . By gentle shaking of the test tube, the presence or absence of red cell agglutination was ascertained macroscopically and microscopically at a magnification of 4 [figures 2 and 3]. Macroscopic observation of the agglutination in dentin (l) and pulp (r) samples microscopic observation of the agglutination in dentin (l, unstained, x100) and pulp (r, unstained, x400) samples (4) data were analyzed in proportion . For comparison, chi - square test or fisher's exact test was used for small sample . Two - tailed p <0.05 was considered as statistically significant, and 0.01 was considered as highly statistically significant . Data were analyzed in proportion . For comparison, chi - square test or fisher's exact test was used for small sample . Two - tailed p <0.05 was considered as statistically significant, and 0.01 was considered as highly statistically significant . Abo blood grouping for dentin and pulp showed a gradual decrease in the sensitivity as the time period increased . In dentine, the sensitivity ranged from 100% to 73% and pulp sensitivity ranged from 100% to 80% . There was no significant difference in the sensitivities of dentin and pulp [table 1]. Abo blood grouping for pulp and dentine rh factor grouping for dentin and pulp showed a gradual decrease in the sensitivity as the time period increased up to 9 months . In dentine, the sensitivity ranged from 100% to 40% and pulp sensitivity ranged from 100% to 23% . Overall pulp showed better sensitivity than dentin except at 12 months time period where dentin showed better sensitivity than pulp . Moreover, in comparison with the sensitivities of dentin and pulp, the p values obtained were not significant [table 2]. Rh factor blood grouping for pulp and dentine when compared the sensitivity of abo and rh factor blood grouping of dentin and pulp in accordance to time period dentin and pulp showed almost similar results from 0 to 9 months . At 12 months both dentin and pulp showed vast difference in the sensitivities of abo and rh blood grouping with significant p value [table 3]. Comparison of abo and rh factor blood grouping for dentin and pulp the overall mean sensitivity of dentin for abo blood grouping is 88% and for rh factor, blood grouping is 79%, with a significant p = 0.03 . The overall mean sensitivity of pulp for abo blood grouping is 90% and for rh factor, blood grouping is 82% with a significant p = 0.04 . Dentin and pulp showed the p = 0.58 for abo blood grouping and 0.46 for rh factor blood grouping [table 4 and figure 4]. Overall comparison of blood group systems and teeth components graph of comparisons of the overall sensitivity of abo and rh factor blood grouping of dentin and pulp lattes have rightly said the fact that belonging to a definite blood group is a fixed character of every human being and can be altered neither by lapse of time nor by intercurrent disease . Human identification is the mainstay of civilization, and the identification of unknown individuals has always been of paramount importance to society . The use of blood group substances in medico - legal examination is grounded on the fact that once a blood group is established in an individual, it remains unchanged for a lifetime . For several decades, the technique ae is the most sensitive and the most widely employed . According to kind, nickolls and periera and outtridge, ae has proved to be markedly more sensitive than the absorption - inhibition test . Ae has shown more success rate than mixed - agglutination for certain antigens . Rh blood group is considered to be the most complex genetically of all the blood type systems since it involves 45 different antigens on the surface of red cell . Teeth are used for blood grouping and are considered as a hallmark for identification of biological materials in forensic investigations . Considering this fact in our present study, an attempt was made to detect abo and rh factor antigen from dentin and pulp over a time period of 0, 3, 6, 9 and 12 months . For abo blood grouping, both dentin and pulp showed 100% sensitivity with the samples tested immediately after extraction and the sensitivity gradually reduced as time period prolonged . Pulp showed better sensitivity than dentin but the difference was statistically insignificant, suggesting both dentin and pulp have almost equal antigenic potential although pulp is better, the sensitivity weakened as the time period increased . Till date, the various studies conducted by smeets et al ., shetty and premlata, ballal and david and ramnarayan et al . At different time period showed pulp to be better tool than dentin and there was a decrease in the sensitivity of the dentin and pulp as the time periods increased . The overall decrease in the sensitivity could be due to dehydration, the loss of pulp antigens, insufficient quantity of pulp, calcification of the canals, cell lysis; contamination of the tooth or time lapse for the procedure . For rh factor blood grouping, both dentin and pulp showed 100% sensitivity when the samples were tested immediately after extraction and the sensitivity gradually reduced as time period prolonged . Pulp showed better results than dentine, but the difference was statistically insignificant, suggesting both dentin and pulp have almost equal antigenic potential which weakened as the time period increased, similar to abo antigens . This may be attributed to autolysis, dehydration and the loss of pulp antigens similar to abo antigens . Is the only study available in english language literature that was conducted for determination of rh factor antigens along with abo blood group antigens in freshly obtained pulp samples . In our study, both pulp and dentin samples were used and tested at 0 month to an extensive time period of 12 months . Abo and rh factor blood grouping for dentin and pulp showed a gradual decrease in the sensitivity as the time period increased . At 12 months, both dentin and pulp showed a drastic decrease in the sensitivity for rh factor blood grouping than the abo blood grouping with a statistically significant difference . The sensitivity of pulp was better than dentin in both the blood grouping systems with an insignificant p value . Abo blood grouping was better than rh factor grouping in both the teeth components and the p values obtained were significant . This indicates that the antigenicity of pulp is better than dentin in both abo and rh blood groups, and abo antigens were better expressed than rh factor blood group antigens . The outcome of our study showed that abo and rh blood group antigens could be detected up to 12 months, and there are no studies in the english literature to compare this study . This shows that pulp contains more amounts of antigen than the dentin and its antigenicity decreases with time . Blood grouping on teeth is not a straight forward technique; the concentrations of blood group antigens are low in the teeth when compared to other tissues and body fluids . In this study, it is assumed that the origin of blood group antigen in dental hard tissue is based on the infusion sedimentation phenomenon combined with inherently present antigens . Considering all the factors that support the presence of blood group antigens in dentin and pulp and also the pitfalls of false positive result or mistyping of blood group, over a period in this study, we came to a close consequence that the results obtained with pulp were better than that of dentine . Another aspect to be highlighted here is abo and rh factor antigens were detected from both dentin and pulp even up to 12 months after extraction of the teeth . Blood grouping has been one of the bases for identification of biological materials in forensic investigations and abo blood grouping is a widely used technique in forensic laboratories . The presence of abo blood group antigens along with rh factor antigens in pulp and hard dental tissues makes it a potential substance in the identification of highly decomposed bodies or body part where teeth and bones are the only significant tissue remains . Blood group substances in the hard dental tissues thus remain unaffected even in adverse environmental conditions . Teeth were used as a mode of identification of blood group in this study because teeth are one of the most indestructible parts of the body and exhibit the least turnover of natural structure . The presence of abo blood group and rh factor antigen in soft and hard tissue makes it possible for identification of the deceased . Ae test to identify blood groups in teeth may be of immense value not only in the identification of accused but also in the investigation of mass disaster and fire victims . On the basis of results obtained in our study, both dentin and pulp are reliable sources of blood group determination for upto 12 months for abo and rh factor blood grouping, especially where teeth ensues to be the only remnants existing for individual identity . Although expression of abo blood groups and rh factor was seen in both dentin and pulp, intensity of abo blood groups and rh factor was higher in pulp than dentin and abo blood group antigens were better expressed than rh factor antigens in both dentin and pulp . Blood group determination from teeth warrants advance exploration as the establishment of identification of a person from the skeletal remains is of paramount importance to a forensic odontologist . Till date, in english language literature no studies have been conducted to detect abo and rh factor blood group antigens from tooth material for up to 12 months . This study detected blood groups antigens of abo and rh systems from tooth material over this extensive time period of 12 months . This study is thus a quantum of what has been known and learned and how much more needs to be understood in this challenging branch of forensic odontology.

Cystic echinococcosis (ce) is a severe zoonosis caused by the cyclophyllidean cestode echinococcus granulosus . The disease has a worldwide distribution, with endemic regions in many countries of the mediterranean basin, north and east africa, western and central asia, china, south america, and australia [1, 2]. Although the distribution of echinococcus granulosus is considered worldwide, it is higher in developing countries in tropics and subtropics, especially in rural communities where there is close contact between dogs and various domestic animals . In some western countries, ce is considered as a reemerging zoonosis due to its resurging prevalence [4, 5]. The worldwide distribution of the disease is partly due to the easy adaptability of the parasite to several domestic and wild intermediate hosts . Clinically, there are three broad morphological forms of echinococcosis that are recognized: cystic echinococcosis caused by e. granulosus, alveolar echinococcosis caused by e. multilocularis, and polycystic echinococcosis caused by e. vogeli and e. oligathrus [79]. The sheep strain (defined as g l on mitochondrial genotypic grounds) is generally considered as the most widespread strain of e. granulosus in the world and the one mainly involved in ce in humans . At least five out of ten strains of e. granulosus strains (g 1 to g 10) have been found to be infective to humans in sub - saharan africa ., disease consequences may include poor quality of life (disability adjusted life years (dalys)), costs of medical treatment, lost opportunity for income generation, and mortality in some cases while in animals there is reduced productivity and monetary losses due to abattoir condemnations of organs [14, 15]. The dalys for human cystic echinococcosis was recently estimated to be more than that for onchocerciasis and almost the same as that for africa trypanosomiasis . The annual ce - associated economic losses on a global basis have been recently estimated to be at least over us$2 billion . In zambia, like in most sub - saharan africa, echinococcosis has been reported in many parts of the country, although not much information is currently available making it one of the neglected tropical diseases . In western province of zambia, hydatid cysts are reported to have been diagnosed from cattle carcasses during meat inspection although most of these reports are inconclusive . However, there has been no comprehensive study carried out thus far to describe echinococcosis infections in both the intermediate and final hosts and also to determine the economic and public health significance . Based on circumstantial evidence, it is assumed that the disease has serious public health and socioeconomic implications given the interactions that exist between cattle, dogs, and humans and also the uncontrolled disposal of abattoir waste and remains from animal slaughters . However, this assertion needed to be supported by well - structured studies . The aim of this study therefore was to determine the prevalence of hydatidosis in cattle presented for slaughter at abattoirs in western province of zambia and assess economic losses due to organ condemnation using a cross - sectional epidemiological survey with the view to identifying intervention measures aimed at reducing transmission of the disease between humans and different animals hosts . The study was conducted in western province of zambia from october 2007 to november 2008 . Western province lies between longitudes 22 degrees and 25 degrees east and latitude 13 degrees 30 mins and 17 degrees 45 mins south . The province covers an area of 126,386 km, which represents about 17% of the total land surface of zambia which covers 752,000 km (figure 1). About 10% (12,950 km) of the total land area consists of a vast sandy upland . The province has a dry and cold winters (april to july), hot and dry season (august to october), and hot and wet summers (november to march). The annual flooding of the zambezi plains controls the pattern of life for the people and livestock in western province with people practice transhumant subsistence livelihood . Thus, during flooding, the largest population of cattle and people are concentrated along the edges of the plains . Western province has a cattle population of approximately 452,400, making it one of the largest cattle producing areas in zambia, while the dog population is estimated at 65,315 with mongu having highest number of dogs at 16,210 followed by kalabo (13,496), shangombo (11,732), sesheke (8,638), kaoma (6,254), senanga (4,750), and lukulu (4,236). Dogs, generally, belong to specific households where feeding is supplemented but often have the freedom to roam and scavenge in the neighbourhood . All cattle are slaughtered within the province, mostly in mongu and senanga abattoirs, due to a movement ban imposed in 1998 as a result of the outbreak of contagious bovine pleural pneumonia (cbpp). Therefore, data obtained from cattle that are slaughtered in mongu is a good representation of the true provincial picture . The study was conducted as a two - tier study involving a prospective abattoir survey and a retrospective review of meat inspection reports at zambeef and starbeef abattoirs in mongu . A retrospective study was carried out based on a review of postmortem reports findings during meat inspection at the abattoirs in the last eleven years (19942007). Data was obtained from district veterinary offices and abattoir reports on meat inspection and movement of livestock carried out in the previous 11 years in western province . Information collected included number of cattle slaughtered, breed and type of organs condemned, number and weight of condemned organs . The aim of this was to provide baseline information and a retrospective understanding of the prevalence, dynamics and spatial distribution of the disease in western province and also to estimate the annual economic loss due to organ condemnation . This study was conducted between october 2007 and october 2008 at zambeef and starbeef abattoirs in mongu district . Cattle that were slaughtered at the two abattoirs were sourced from all the seven districts of western province except sesheke district . All 4061 cattle that were slaughtered during the study period were included in the survey . The slaughtered cattle were subjected to thorough postmortem inspection and lesioned organs were identified and samples were collected . Prior to commencement of the prospective study, meat inspectors at the two slaughterhouses underwent an in - house refresher training in recognition of hydatid cysts in various organs of the carcasses according to the procedures recommended by fao / unep / who (1994). Each animal that was slaughtered was uniquely identified using stock movement permits that included the veterinary camp of origin in the district and further information was obtained by interviewing the owner . The age of the animals was obtained by interviewing the owners in cases where the ultimate owner brought the cattle; otherwise the age was estimated using dentition as described by jenkins . Visceral organs including lungs, liver, heart, spleen, and kidneys were examined, through visual observation, palpation and systematic incision in each carcass according to procedures recommended by fao / unep / who (1994). Hydatid cysts where identified through visual inspection and palpation of organs during meat inspection and enumerated . A sample of hydatid cysts during inspection was removed whole and collected in polythene bags . A separate polythene bag was used for hydatid cysts obtained from one animal and was uniquely labelled and stored in ice before transportation, within one hour, to mongu regional laboratory for viability determination . Cattle were classified as positive for hydatidosis if it was found with one or more hydatid cysts in any of the internal organs . At mongu regional laboratory, the collected cysts were individually grossly examined for degeneration and calcification as described by oostburg et al . . The cyst wall was carefully incised with a scalpel blade and the contents poured into a petri dish . The contents were examined under a microscope (40x magnification) for the presence of protoscoleces . The germinal layer was also put in glycerine and placed between two microscopic glass slides and examined for the presence of protoscoleces . Cysts that did not contain protoscoleces contained pus or were calcified were considered as sterile or not viable . Further, the viability of the protoscolices was checked under the microscope using the dye exclusion principle after staining with 0.1% eosin stain for 15 minutes . The protoscolices that took up the stain were classified as dead while those that did not were considered to be alive and viable [18, 19]. The loss attributed to condemnations of offal due to echinococcus was determined using a modification of the formula as described by yamene (1990) cited by getaw et al . . This was on the basis of the average price of wholesome and intact visceral organs obtained from zambeef and starbeef abattoirs mongu . Data was stored in microsoft excel spread and transferred to stata statistical packages version 10 (stata corp . Infections in cattle for both prospective and retrospective data was determined as proportion of the test - positive subjects against the total number tested . Apparent prevalence estimates were converted into true prevalence values by taking into account the sensitivity and specificity of the test methods as described in dohoo (2003). The annual economic loss as a result of condemned organ was estimated by taking into account the average number of cattle slaughtered per annum at the zambeef and starbeef abattoir and the percentage of condemned organs using the following formula described by yemane (1990) as cited by getaw et al . . (1)annual loss=(npsiluclu)+(npsilicli)+(npsiheche)+(npsikicki), where nps: total number of positive animal slaughter, ilu: prevalence of lung hydatidosis, ili: prevalence of liver hydatidosis, ihe: prevalence of heart hydatidosis, iki: prevalence of kidney hydatidosis, clu: cost of lung, cli: cost of liver, che: cost of heart, and cki: cost of kidney . A retrospective study was carried out based on a review of postmortem report findings during meat inspection at the abattoirs over a period of eleven years from 1994 to 2007 (with exemption of 1997, 1998, and 2002 where data was missing). During this period, 158, 456 bovines were slaughtered and inspected, and 4689 cases of bovine hydatidosis were recorded (table 2). The overall combined prevalence of bovine echinococcosis during the period under review was estimated at 3.0% (table 1) which was close to prevalence observed in our prospective study . Annual prevalence ranged from the lowest at 1.56% (n = 12641) in 2006 to the highest at 4.7% (n = 2633) in 2001 . A review of the postmortem records over an eleven year period revealed that the distribution of hydatid cysts in bovine was highest in lung at 93.47% (95% ci: 92.7594.14) followed by liver at 6.55% (95% ci: 5.887.29) and spleen with the lowest at 0.02% (95% ci: 0.000.12) prevalence . A total of 4061 cattle from mongu (n = 2441), senanga (n = 577), kalabo (n = 653), lukulu (n = 335), shangombo (n = 47), and kaoma (n = 8) were slaughtered at the zambeef and starbeef abattoirs between october 2007 and november 2008 . Out of this, 84 (2.1%) carcasses (table 3) were diagnosed positive for hydatidosis during postmortem inspections . There was variation in prevalence of hydatidosis according to the district of origin, where cattle coming from mongu had the highest prevalence of cyst positive cases (2.5%) compared to senanga (2.1%), kalabo (1.4%), and lukulu (0.6%) (table 3). Sex was found to be positively associated with hydatidosis (p = 0.035) with female cattle being more likely to test positive than males (odds ratio = 1.62). On the other hand, hydatidosis was independent of age (p = 0.31) where the mean age among the positives was 7.8 years (range: 7.47.6) and that among the negatives was 7.5 years (range: 7.38.3). In terms of distribution of hydatid cysts by organ, 51.2% were found in lungs, 47.6% were in livers, while 1.2% were in the kidneys . Mukukutu camp in senanga district accounted for the highest prevalence at 4.0% (95% ci 3.811.8%), while lukulu central camp in lukulu district had the lowest prevalence at 0.3% (95% ci 0.20.9%). On comparison of camps in different districts, it was observed that in mongu, the highest prevalence of bovine hydatidosis was in limulunga veterinary camp at 2.9% (95% ci 1.44.4%) with the lowest prevalence in luandui camp at 1.5% (95% ci 0.53.7%). In senanga district, the highest prevalence was in mukukutu camp at 4.0% (95% ci 3.811.8%) and the lowest was mouyo camp at 1.6% (95% ci 0.043.2%). In kalabo district, the highest prevalence was observed in sikongo camp at 3.3% (95% ci 1.28.0%). In lukulu district, the highest prevalence was in mbanga camp at 1.8% (95% ci 1.75.3%) and lowest in lukulu central camp at 0.3% (95% ci 0.20.9%). The overall median number of cysts in an organ was 6 (range 221), in the lungs the median was 6 (range 221), and liver the median was 4 (range; 35). The number of hydatid cysts that were examined in the lung was 108 while in the liver it was 16 . The lung had a highest density of cysts per organ compared to the liver (table 4). There was no significant difference in viability rate of hydatid cysts recovered from the lung (43.5%) and in liver (43.8%). The prices used in the estimation of annual economic loss from condemned organs, were the 2011 average prices for wholesome and intact visceral organs obtained from zambeef butchery in mongu . While the average weights of the various organs were calculated from the data obtained from the abattoir prospective study . The average weight of a lung was estimated at 2.92 kg, liver and spleen were 3.34 kg and 2.00 kg, respectively . The average cost of lung was zmk (zambian kwacha) 12,000 per kg, liver zmk 18,000 per kg, and spleen zmk 12,000 . The cost of one lung = average weight cost / kg (2.92 @ 12000) = zmk 35,040; cost of liver = average weight cost / kg (3.34 @ 18000) = zmk 60,120; cost of spleen= average weight cost / kg (2 @ 12000) = zmk24, 000, average annual slaughter= 14,405 . In this study, we investigated the prevalence of hydatidosis based on pm findings at two abattoirs in western province of zambia . It is therefore noted that the prevalence estimates provided here may have some bias as abattoir sample populations is not always representative of the reference populations where animals are drawn . This is often so because animals brought for slaughters are those that are old or out of production . Considering the reduced sensitivity of pm inspection - based diagnosis, there is always a possibility that some positive cases were missed resulting in underreporting the actual disease burden . Despite these short - comings, abattoir survey data is routinely used to estimate disease burden because of easy feasibility of conducting abattoir surveys compared to field surveys based on random study designs . Besides, abattoir data provides opportunity for developing intervention strategies by timely diagnosis and condemning carcasses infected with zoonoses likely to enter the food chain . The observed prevalence of hydatid cysts in cattle sampled at the two abattoirs in mongu was found to be low (2.1%) and was comparable to that observed during the retrospective survey (3.0%). Furthermore, the findings in this study were in agreement with that observed in a study done in sudan which reported a prevalence of 3% in cattle . In arusha tanzania, a study by nonga and karimuribo reported a prevalence of 4.2% in cattle . Similarly, for instance, rkia azlaf and allal dakkak reported prevalence of 23.0% bovine hydatidosis in morocco and so did kebede in ethiopia who reported a prevalence of 22.1% . In our study, the distribution of hydatidosis varied according to district with mongu reporting the highest prevalence compared to other districts . The reason for the high prevalence in mongu could be attributed to a high numbers of cattle and dog populations coupled with a high number of home slaughters during ceremonies, which in some cases are not inspected by the veterinary department staff . There is an increased dog and cattle interaction due to high populations and free range rearing of cattle which are often herded by boys with dogs; this increases contact of cattle with dog faeces . Further, dog access to slaughterhouse waste in mongu abattoirs is likely to increase exposure of both cattle and dogs in the district . Sex was found to be positively associated with hydatidosis (p = 0.035) with female cattle being more likely to test positive than males ., in iran, who observed that the prevalence was higher in females than males . There was a significant difference in the prevalence of hydatid cysts between carcasses slaughtered in 2007 and 2008 (p = 0.024), where prevalence of bovine hydatidosis in 2007 was 1.3% (95% ci: 0.691.93) and in 2008 was 2.4% (95% ci: 1.82.9). This could be as a result of more animals coming from areas of higher prevalence of bovine hydatidosis such as mongu and senanga in 2008 than in 2007 . However, this could not be fully ascertained due to absence of trace back information during the period under review . The lung was found to be the most affected organ (51.2%) compared to the liver (47.6%) and the kidney (1.2%). This is in agreement with what was reported by getaw et al . Who observed that the lung had a higher prevalence at 55.2% and the liver at 37.1% while the kidney was the least affacted organ . The results are also in agreement with findings by cadmus and adesokan (2009) in nigeria, and kebede et al . Who did their study in ethiopia . However, our results are at odds with the findings from a study conducted in libya where researchers reported higher prevalence in the liver than in the lung and al - khalid (1998) cited by dakkak who showed that in libya, 75% of the positive bovine hydatidosis cases were in the liver and 37.5% in the lung and 12.5% in the spleen . The reason why the lung and liver are mostly affected could be due to the fact that the lungs and livers are the first capillary beds encountered by migrating echinococcus oncospheres via the portal vein route before any other peripheral organs . The lungs however have a larger capillary bed than any other organs and this could account for the observed higher prevalence than seen in the other organs . In humans, however, the liver is most commonly affected . The explanation to this differences in the predirection sites between cattle and human is beyond the scope of this study . Cysts viability study revealed that the overall percentage of viable cyst in this study was 43.5% which is comparable to findings by other researchers like ibrahim who found cyst viability of 47.8% in sheep and 24% in goats ., who did not observe any viable cyst from their survey and berhe who found a lower viability rate of 10.7% in cattle in tigray region of ethiopia . The possible reason why no viable cysts were observed by rinaldi et al . Could be due to the differences in immunological responses by different individual hosts or deworming of the animals by use of antihelmintics . Out of a total of 19 hydatid cyst infested organs that were investigated (15 lungs and 4 livers) for cyst fertility, viability, and density, it was found that the lung had a higher average density of cysts infestation (7 cysts per lung) (table 4) while the liver had a low hydatid cyst density (4 cysts per liver). This was however different from findings by ibrahim in saudi arabia who observed that the liver had a higher cyst density . The difference in the cyst density could mainly be attributed to the higher vascularisation of lung tissue compared to liver . The other reason in the difference in cyst density could be as a result of the soft texture of the lung tissue in comparison to liver, which has a harder texture thus restricting hydatid cyst development . The number of hydatid cysts that were examined in the lung was 108 while in the liver was 16 . Most of the dead cysts in the liver were found to be calcified compared to the lung ., kebede et al ., and berhe who reported a higher fertility rate of pulmonary and lower fertility rate in hepatic cysts . This could probably be due to the various metabolic reactions that take place in the liver as compared to lungs . However ibrahim found a higher fertility rate in liver at 38.8% than in the lung at 25.1% and so did dalimi et al . Who reported a higher fertility rate in the liver than in the lung . The high percentage of viable cysts indicates that there is a high risk of dog exposure in situations where offal are carelessly disposed of and dogs have access to the infected offal, like the situation is in western province of zambia . This points to a possible intervention area in which dogs should be prevented from ingesting infected with cysts such as the lungs and liver . In this study, the annual economic loss as a result of condemnation of organs due to bovine hydatidosis was low at k 15, 894,039.00. (3,311 us$) per annum . The loss was found to be low due to the low prevalence of hydatidosis in cattle in western province of zambia . The total annual loss could be greater than the estimated amount bearing in mind that this only took into account direct losses and not indirect losses as a result of weight loss due to ce and other losses such as reduced milk production and reduction in reproduction in cattle . The main thrust of the study was to describe the hydatidosis situation in western province so as to increase public health awareness, describe the socioeconomic impact, and to recommend possible mitigation measures . It should be noted however, that echinococcosis is a disease of multiple hosts and the objective of this study could only be addressed by the application of conventional observational studies . This study has demonstrated that hydatidosis is an important disease and is endemic in western province . The disease also causes considerable economic losses as a result of offal condemnations . Despite the low infection rate demonstrated by the current study, there are certain socioeconomic conditions that are favourable for the existence of ce, and therefore ce still remains one of the most important helminth zoonotic disease hence the need for increased attention in control and prevention of the disease . Livestock and dog echinococcosis prevalence studies and surveillance can help map out ce risk landscape profiles that will determine communities at greatest risk to human ce . A molecular analysis of human and animal hydatidosis would be desirable in order to effectively map out epidemiology of the disease and determine the spread of the disease . A specific study of the disease in dogs could also help in knowing the prevalence in the definitive host . Wildlife species have not been shown to harbour e. granulosus in zambia . In view of the extensive livestock / wildlife interface areas in the province, furthermore, a study in small ruminants such as sheep and goats may improve epidemiological understanding of the disease in zambia . With the current study having been done on cattle, it is also suggested that in areas where there is a presence of large numbers of wildlife definitive hosts such as observed in some parts of kalabo, lukulu, and kaoma districts, increased effort should be made to sample some of the possible wildlife definitive host of echinococcus spp . Effort should be made to ascertain the host specificity of local strains of the parasite in respect to cattle and other domestic animals . To effectively come up with a control program, possible wildlife reservoirs, and the survival of eggs under the local climatic and soil conditions have to be investigated.

Septic internal jugular vein - sigmoid sinus thrombosis is a rare condition that complicates local and regional infectious inflammatory processes occurring in the head and neck . These include deep neck infections, lemierre syndrome, and central venous catheterization (cvc) or cannulation17). This is a life - threatening condition and prompt management is essential to decrease the potential for thrombosis - related morbidity and mortality . We report a case of internal jugular vein - sigmoid sinus thrombosis due to a misplaced central venous catheter . A 52-year - old woman presented with a severe bursting headache, vomiting, and a drowsy mentality . Brain computed tomography (ct) scan revealed subarachnoid hemorrhage in the basal cistern with a small amount of hematoma at the left sylvian fissure (fig . We identified an aneurysmal rupture of the middle cerebral artery on the left side by cerebral catheter angiography (fig . Postoperative chest x - ray was normal except for malposition of the central venous catheter in the right internal jugular vein (fig . Ten days later, the patient developed a fever, elevated white blood cell count to 227000, and elevated c - reactive protein to 33.6 . The central venous catheter was removed, and antibiotics were administrated through a different intravenous route . Despite these managements, the patient was stupor the next day . Brain ct scan revealed cerebral infarction with hemorrhagic transformation in the right temporal lobe, and ct angiography did not identify vasospasm (fig . Retrospective analysis of the contrast - enhanced ct scan and ct angiography showed an " empty delta sign " and absence of venous flow within the right internal jugular vein - sigmoid sinus that was sufficient for diagnosis of sinus thrombosis (fig . Results of central venous catheter tip and blood culture was reported staphylococcus epidermidis and methicillin - resistant staphylococcus aureus, respectively . We could not start systemic heparinization due to hemorrhagic transformation of the cerebral infarction in the right temporal lobe . We administrated mannitol and steroids to manage increased intracranial pressure; however, the patient died from severe pneumonia due to septic emboli after one week (fig . 2c). Indications for a cvc are the intravenous administration of drugs, parenteral nutrition, hemodialysis, and hemodynamic monitoring13). At many institutions, cvc is routinely inserted before undergoing major surgery or treating patients with critical illnesses or cancer . The most frequently available anatomical sites for cvc are the subclavian and internal jugular veins . These procedures carry a substantial risk of mechanical lesions, such as arterial puncture, pneumothorax, cardiac tamponade, nerve lesions, or thrombotic or septic complications13). Malpositioning of the catheter tip may happen more often in the subclavian vein than the internal jugular vein . The reported incidence of primary misplacement of the catheter tip after infraclavicular subclavian vein catheterization varies from 5% to 24% even when inserted by experienced clinicians7). Inadvertent catheterization of the ipsilateral internal jugular vein is one of the most common misplacements, with a reported incidence of around 7%15). The positioning of catheter tips within the cardiac silhouette is associated with increased risk of cardiac tamponade6). Also, positioning of the catheter tip in the subclavian vein is associated with a high risk of thrombus formation and vessel occlusion2). The risk of thrombosis may increase when hyperosmolar parenteral nutrition fluid is administered through a misplaced central venous catheter into a internal jugular vein3,15). Moreover, malpositioned catheter tips can damage the endothelium and precipitate the formation of thrombi17). When a cvc causes thrombosis, the risk of catheter - related sepsis may increase . In patients with a cvc, the risk of catheter - related infection was reported to range between 1% and 10%1). Contamination of a thrombus from the skin puncture site may result in septic endophlebitis, and occasional blood - borne infections may also contaminate the thrombus . Embolic septic thrombi may involve the lungs and, less frequently, the joints, viscera, and brain17). Subclavian or internal jugular vein thrombosis associated with indwelling catheters will propagate into other vessels, but extension into the intracranial sinuses and veins is rare . Three reports describe an association between cerebral venous sinus thrombosis and central venous hyperalimentation due to placement of the catheter tip in the internal jugular vein3,15,16). The authors warn of the potential for thrombosis due to retrograde infusion into the valveless internal jugular - dural sinus system but also suggest that the small caliber of the vein may predispose to thrombosis . In our case, we found the malpositioned catheter tip in the internal jugular vein on follow - up chest x - ray, but it was ignored . All intravenous fluids (e.g., total parenteral nutrition, mannitol, antibiotics) were administered through the misplaced catheter . The patient's condition worsened, leading to thrombosis of the internal jugular vein secondary to sigmoid sinus thrombosis . Since the advent of antibiotics fever, chills, otalgia, tenderness to percussion over the mastoid emissary vein, headache and vomiting are common but not pathognomonic features8,18). Occasionally, neurologic symptoms are related to increased intracranial pressure or infarct and present as deteriorating mental status, lethargy, seizures, hemiplegia, and coma, and may lead to death . Rarely, remote septic conditions such as pneumonia are the presenting symptoms9). Because of the nonspecific signs and symptoms of disease and the masking effects of antibiotics, diagnosis is difficult . The diagnosis of sigmoid sinus thrombosis can be confirmed by ct, magnetic resonance imaging, or angiography . Contrast - enhanced ct scans demonstrate multiple intraluminal filling defects and nonvisualization of the sinus18). In addition, low density lumen, sharply defined dense vessel wall, or distension of the thrombosed vein, such as the " empty delta sign ", are positive signs for sinus thrombosis19). Vascular imaging with cerebral angiography is highly specific for the recognition of sinus thrombosis because it can detect the lack of blood flow in thrombosed cerebral veins and dural sinuses . Chest x - rays may also demonstrate septic embolic pleura - pulmonary complications, which are often bilateral, by revealing nodular infiltrates with pleural effusion17). Retrospectively, we confirmed that contrast - enhanced brain ct scans revealed a low density lumen surrounding a sharply enhanced dense vessel wall at the sigmoid sinus on the right side . Also, the right internal jugular vein and sigmoid sinus were not visualized on ct angiography . Treatment consists of aggressive antimicrobial therapy, heparinization, anticoagulation, and decreasing intracranial pressure . Antibiotic selections is directed toward the causative pathogen cultured at the initial site of infection . Recent studies have shown heparinization to be safe and beneficial, despite the possibility of an increased risk of hemorrhage4,10,16). Currently, heparin is recommended as the initial drug of choice for cerebral venous sinus thrombosis followed by long - term anticoagulation with warfarin5,10,12). Although these procedures allow for rapid clot removal and reduction of venous hypertension, hemorrhagic complications can occur, leading to be high morbidity and mortality14). Occlusion of the cerebral veins due to thrombosis may induce localized brain edema and venous infarction resulting in elevated intracranial pressure . Finally, death frequently results from increased intracranial pressure caused by obstruction of venous and cerebrospinal outflow11). In our case culture results of the subclavian catheter tip and blood were confirmed as staphylococcus epidermidis and methicillin - resistant staphylococcus aureus, respectively, and we switched from broad - spectrum antibiotics to vancomycin . Unfortunately, we could not start heparinization due to concurrent cerebral hemorrhage of being transformed from cerebral infarction . Severe pneumonia due to septic emboli eventually developed in both lung fields, and the patient died . Diagnosis of internal jugular vein - sigmoid sinus thrombosis is challenging due to vague clinical features . Therefore, if patients with malpositioned cvcs present with symptoms of fever, chills, headache, vomiting, increased intracranial pressure, mental deterioration, and focal neurologic deficits, intracranial sinus thrombosis should be considered . Even vague symptoms may need to be investigated radiologically to discover a thrombosis early, when it can be treated.

They are also used to treat multiple myeloma, bone metastases, and calcium disorders 2 . These patients reportedly often develop bisphosphonaterelated osteonecrosis of the jaw (bronj) that is caused by subsequent dental surgery 3, 4 . In general, it is thought that nitrogencontaining bisphosphonates are much high risk of osteonecrosis than nitrogen notcontaining bisphosphonates . Thus, medications for cancer, such as zoledronate (zometa), are thought to have much high risk of osteonecrosis than medications for osteoporosis, such as alendronate (bonalon) 5 . Besides bisphosphonates, other antiresorptive agents such as denosumab and antiangiogenic medications also cause osteonecrosis 6 . Thus, the american association of oral and maxillofacial surgeons position paper published in 2014 and the committee recommended changing the nomenclature of bronj to the term medicationrelated osteonecrosis of the jaw (mronj) to accommodate the growing number of jaw osteonecrosis cases associated with other antiresorptive and antiangiogenic therapies 7 . According to the position paper, there are three categories of risk factors for mronj: (1) medicationrelated risk factors (e.g., bisphosphonate use), (2) local factors (e.g., dental surgery), and (3) demographic and systemic factors and other medication factors . In this case report, the patient developed mronj and died of sepsis, although he received shortterm oral bisphosphonate therapy and had not received any dental surgical treatment because he was completely edentulous . However, he had many systemic factors such as diabetes, cirrhosis, and steroid use for interstitial pneumonia . The presence of multiple systemic factors can be a very high risk for mronj, even though there are few medicationrelated factors and local factors 8, 9, 10 . The patient was a 59yearold man with a history of smoking and drinking, insulin use for diabetes, cirrhosis associated with chronic hepatitis c, steroid use for interstitial pneumonia, sepsis, and spinal disk herniation . This study was conducted according to the guidelines of hiroshima city hiroshima citizens hospital, and a written informed consent was obtained from this patient . White blood cell count (wbc) was 7800/l and creactive protein (crp) level was 15.523 he was treated with intravenous steroids (i.e., methylprednisolone sodium succinate 1000 mg / day for 3 days, prednisolone sodium succinate 20 mg twice daily for 4 days, and methylprednisolone sodium succinate 1000 mg / day for 3 days). This regimen was followed by oral steroids (i.e., prednisolone 60 mg / day for 2 months). To prevent steroidinduced osteoporosis, he was treated with oral bisphosphonate (i.e., alendronate 35 mg / week for 7 weeks). However, the administration of these drugs was stopped because of a right buccal space infection . The patient had severe swelling from the right buccal area to the infraorbital region, hypesthesia, and tenderness, but no spontaneous pain (fig . There were ulcerations and exposed bone on the alveolar part of the right incisor and molar (fig . Computer tomography (ct) scanning with a radiocontrast agent showed swelling in the right buccal area, but showed no mandibular bone resorption (fig . 2b). Magnetic resonance imaging (mri) demonstrated an abnormal signal (e.g., low signal on t1weighted [t1w1] mri and high signal on t2weighted [t2w1] mri) in the bone marrow of the right mandibular angle (fig . Based on these results, the patient was diagnosed as having right mandibular cellulitis, sepsis, and disseminated intravascular coagulation (dic). We administered intravenous antibiotic treatment and irrigated part of the exposed bone with povidone iodine every weekday . At 3 days after the first visit, sequestrectomy and drainage therefore, we changed the antibiotics from meropenem hydrate (meropen; sumitomo dainippon pharma co., ltd ., osaka, japan) alone to meropenem hydrate and ampicillin / sulbactam (unasyn s; pfizer inc . One month after the first visit, swelling from right buccal area had nearly disappeared (fig . 4b). Exposed bone remained on the alveolar portion of the right mandibular molar and pus discharge was present (fig . Antibioticresistant bacteria (grampositive bacilli) were detected at that time (table 1). Therefore, we changed the antibiotics to sitafloxacin (gracevit; daiichi sankyo, tokyo, japan) (fig . 3). Seven weeks after the first visit, his condition had taken a turn for the worse ., he died of multiorgan failure . Finally, we diagnosed this patient as mronj . His multiple systematic factors and mronj caused lethal sepsis because it fulfilled the diagnostic criteria: previous treatment with bisphosphonate, exposed bone in the maxillofacial region that has persisted for 8 weeks and no history of radiation therapy to the jaws . (b) bisphosphonaterelated exposed necrotic bone in the right posterior mandibular (during drainage). (a) no bone resorption is present on the right part of the mandibula in orthopantomogram analysis . (b, c) right buccal swelling is apparent, but there is no bone resorption of the mandibula, based on computer tomography (ct) analysis . (d, e) abnormal signal (i.e., a low signal on t1weighted imaging [tiwi] and a high signal on t2weighted imaging [t2w1]) of the bone marrow on the right angle part of the mandibula . Bacterial identification and microbial sensitivity test the transition in the laboratory test values for the white blood cell count (wbc) and the creactive protein (crp) level, and the use of several antibiotics for cellulitis and medicationrelated osteonecrosis of the jaws (mronj). (a) disappearance of swelling from the right buccal area to the infraorbital region . (c) discharge of pus by exposed bone on the alveolar part of the right mandibular molar . In recently years, there have been many reports on osteonecrosis of the jaw (onj) caused by bisphosphonates and by other antiresorptive and antiangiogenic therapies 6, 11, 12 . Bisphosphonaterelated osteonecrosis of the jaw to medicationrelated osteonecrosis of the jaw 7 . Denosumab, a rank ligand inhibitor, is an antiresorptive agent and used to treat osteoporosis, multiple myeloma, and giant cell tumor 13 . The manufacturer reports that the frequency of onj with denosumab is nearly the same as the frequency with zoledronic acid treatment 14 . Thus, it may be that the development of onj is associated with the inhibition of bone resorption, rather than the use of a certain type of drug . However, it is considered that careful attention is required for the use of bisphosphonates in the present case due to the frequent use for the treatment of osteoporosis, osteopenia, and other diseases . Many reports indicate that the incidence of onj is significantly higher with the use of intravenous (iv) bisphosphonates such as zoledronic acid (zometa; novartis, basel, switzerland) than by the use of oral bisphosphonate 3, 15 . The incidence of onj because of the use oral bisphosphonates was higher in japan than in europe and america 16 . The period of drug use is also important, and longterm use is considered a high risk for the development of onj . In our patient, the period of oral bisphosphonates use was only 7 weeks, but it caused severe symptoms . It may be that these symptoms were because of his multiple systemic factors . In this case thus, it is thought that there were another option not to use bisphosphonate for such a highrisk patient or at least before administration we had to perform a risk assessment for mronj enough . In addition, the presence of comorbid condition such as obesity, alcohol use, and smoking was a risk factor for mronj 9 . Poor oral hygiene and oral infection such as periodontal disease are also risk factors 18, 19 . The dentures were possibly contaminated with oral bacteria . In this case, we do not know whether the dentures were illfitting and there were denturerelated traumatic ulcers because he already had ulcerations and exposed bone when we first saw him . However, we think denturerelated traumatic ulcers might have promoted a mronj . Osteonecrosis of the jaw has also been reported in patients with no history of surgery or in edentulous regions of the jaw 20 . Physicians have to be careful with the use of bisphosphonates or other bone resorption inhibitors in patients, regardless of the presence or absence of teeth or surgical history . The most important thing to prevent mronj it required cooperation among physicians, nurses, dentists, dental hygienists, pharmacists, and other medical staff 7 . Physicians who prescribe bisphosphonates or other bone resorption inhibitors have to provide a detailed explanation to patients of the risk of mronj and consult dentists if a patient has even a small risk factor . To prevent mronj, dentists and dental hygienists have to explain sufficiently to patients the importance of oral hygiene, and perform oral care and dental treatments before patients use these drugs . There is a tendency to believe that mronj is caused by longterm bisphosphonate use (especially intravenous bisphosphonate) and dental surgery . We found that multiple systemic factors can be a worse risk factor for mronj because it may cause lethal disease such as sepsis . Physicians have to be careful when administering bisphosphonate, other antiresorptive agents, and antiangiogenic medications for patients with multiple systemic factors, even if the patients seem to have no problems in the oral cavity . Ky: involved in study conception, design, and drafting manuscript; as: involved in study conception, design, and is the chief doctor for the patient; fo: is one of the main doctors for the patient and performed data acquisition; mn: is supervisor and main doctor for the patient; ks and ey: performed analysis and interpretation of data; yh: is one of the main doctors for the patient and performed data acquisition; si: made critical revision for this report; st: made critical revision and is the corresponding author.

Supported by nci 2p50 ca09825806, nci u01 ca168394, stand up to cancer / aacr dream team translational cancer research grant su2c - aacr - dt0209, tcga gdac grant (nih / nci u24 ca143883) to gbm; mdacc uterine spore career development award (nci p50ca098258) to lwt.

To review the current knowledge about nonpharmacologic approaches in the prevention and early treatment of type 2 diabetes . This study reviewed the research reports dealing with nonpharmacologic interventions aimed at preventing type 2 diabetes with early lifestyle interventions . The results from the randomized controlled trials all show that people with impaired glucose tolerance who received enhanced lifestyle advice had significantly lower (on average 50% reduced) incidence of type 2 diabetes compared with those allocated to receive usual care . Individuals who were able to correct their lifestyle habits as recommended for usual healthy life patterns were mostly protected against type 2 diabetes . Thus, compelling evidence exists that most of the cases of type 2 diabetes can be prevented or at least the onset of the disease can be significantly delayed . Randomized controlled trials have unequivocally demonstrated that lifestyle management is highly efficient in the prevention and also in the early management of type 2 diabetes . This evidence of lifestyle modification in diabetes prevention is stronger than for most other multifactorial diseases . In the early randomized intervention study in malmhus, sweden (19), lower rates of type 2 diabetes was found in igt men randomized to dietary intervention compared with those who received no therapy . More recently, several trials have tested the efficacy of lifestyle intervention in prevention of type 2 diabetes . The feasibility of diet and exercise intervention in men with igt was assessed in another study in malm, sweden (20). Because the reference group comprised of men who did not want to join the intervention, the groups were not randomly assigned . The lifestyle intervention aimed at reducing the intake of refined sugar, simple carbohydrates, fat, saturated fat, energy, and alcohol and an increase in the intake of complex carbohydrates and vegetables . Physical activity training consisted of two weekly 60-min sessions with various dynamic activities . By the end of the 5-year study period, 11 and 29% of the men in the intervention group and reference group had developed type 2 diabetes, respectively . Overall, the progression to diabetes in these swedish men was relatively low, even in the reference group compared with the data from the observational studies (1). The intervention resulted in significant changes in lifestyle and physiological parameters . In another study, 577 subjects with igt were assigned either to a control, exercise alone, diet alone, or exercise plus diet group in da - qing, china (14), using a cluster - randomized trial design . Participants were assigned to clinics for dietary intervention and were encouraged to reduce weight if bmi was 25 kg / m (61% of all participants) aiming at 23 kg / m; otherwise high - carbohydrate (5565% of energy) and moderate - fat (2530% of energy) diet was recommended . The participants were encouraged to increase their level of leisure - time physical activity by at least 12 units per day in clinics assigned to exercise intervention . One unit would correspond for instance to 30 min slow walking, 10 min slow running, or 5 min swimming . The cumulative 6-year incidence of type 2 diabetes was lower in each of the three intervention groups (4146%) compared with 68% in the control group . The results of the finnish diabetes prevention study (dps) provided the first convincing evidence from a proper randomized controlled trial that type 2 diabetes can be prevented by lifestyle modification (21). A total of 522 individuals with igt were randomized to either an intensive lifestyle or a control intervention: during an average of 3.2 years of follow - up, type 2 diabetes incidence was reduced by 58% in the lifestyle group . The lifestyle intervention goals were 1) reduction in weight of 5%, 2) total fat intake <30% of energy, 3) saturated fat intake <10% of energy, 4) fiber intake 15 g/1,000 kcal, and 5) moderate exercise for 30 min / day . During the first year of the study, body weight decreased on average 4.5 kg in the intervention group and 1.0 kg in the control group subjects (p <0.0001). Indicators of central adiposity and fasting glucose and insulin, 2-h postchallenge glucose and insulin, and a1c were all reduced significantly in the intervention group compared with the control group at 1-year examination (fig . Changes in clinical and metabolic characteristics among the intervention and control group participants of the dps . 2h - p - gluc, 2-h plasma glucose; diast, diastolic blood pressure; f - p - gluc, fasting plasma glucose; s, serum; syst, systolic blood pressure . Diabetes prevention program (dpp) (22) recruited 3,234 individuals with igt (and fasting plasma glucose 95 mg / dl) who were randomized to receive intensive dietary and exercise counseling, metformin, or placebo . The main aims of the intervention were 7% weight reduction and 150 min / week moderate physical activity . The relative risk reduction after 2.8 years was 58% in the lifestyle intervention group compared with the placebo group . The effect of lifestyle was higher than the effect of metformin, which showed 35% relative risk reduction . During the first year of the intervention, weight reduction was 5.6 kg (6%), with slight, gradual regain to the end of the study at year 4 (23). The indian diabetes prevention program (14) recruited 531 people with igt who were randomized into four groups (control, lifestyle modification, metformin, and combined lifestyle modification and metformin). Lifestyle modification included advice on physical activity (30 min of brisk walking per day) and reduction in total calories, refined carbohydrates and fats, avoidance of sugar, and increase in fiber - rich foods . After median follow - up of 30 months, the relative risk reduction in type 2 diabetes incidence was with lifestyle modification, 26.4% with metformin, and 28.2% with lifestyle modification and metformin, as compared with the control group . Thus, there was no added benefit from combining the pharmacologic and lifestyle interventions . The japanese trial (24) included 458 igt men randomized to receive either intensive lifestyle intervention (n = 102) or standard intervention (n = 356). The aims of the intensive intervention were body weight reduction if bmi was 22 kg / m (otherwise, to maintain present weight), to consume large amounts of vegetables while reducing the amount of other foods by 10%, reduction of fat (<50 g / day) and alcohol intake (<50 g / day), and physical activity> 3040 min / day . The cumulative 4-year incidence of type 2 diabetes in the intervention group was 67% lower than in the control group . Body weight decreased by 2.2 and 0.4 kg in the intervention and control groups, respectively . The trials listed above have demonstrated the benefits of healthy lifestyle on delaying the deterioration of glucose tolerance to manifest type 2 diabetes, at least as long as the intervention continued . Data on possible long - term effects of such active lifestyle counseling are scarce . The 12-year follow - up of the malm study (25) revealed that mortality among men in the former igt intervention group was lower than in the control group (6.5 vs. 14.0/1,000 person - years, p = 0.009). In a median 7-year follow - up of the dps, the marked reduction in type 2 diabetes incidence was sustained (13). More importantly, after a median postintervention follow - up of 3 years, type 2 diabetes incidence was 4.6 and 7.2 per 100 person - years in the intervention and control groups, respectively (log - rank test, p = 0.0401), i.e., a 36% additional risk reduction . The absolute risk difference between groups increased during the postintervention period: intensive lifestyle intervention for a limited time can yield long - term benefits on type 2 diabetes risk in individuals with igt . The 20-year follow - up of the original da qing cohort showed that a lower type 2 diabetes incidence persisted in the lifestyle intervention groups (combined) compared with control participants . The risk reduction remained essentially the same also during the postintervention period (26). (26) observed no statistically significant differences in cvd events, cvd, or total mortality between the control group and the combined intervention groups, but cvd mortality tended to be lower (17%) among individuals who had received lifestyle intervention . In most of the published prevention trials, the main aim was to see if comprehensive lifestyle intervention reduces type 2 diabetes risk . In the chinese prevention study (14), an attempt to determine whether diet or exercise intervention is more effective by randomizing the participating clinics to diet only, physical activity only, or diet plus physical activity intervention revealed no difference in outcome between the two interventions . In the dps, the risk of being diagnosed with diabetes was strongly associated with the number of lifestyle goals achieved (21). Success in achieving the intervention goals in the dps was estimated from the food records and exercise questionnaires . The success score (from 0 to 5) there was a strong inverse correlation between the success score and the incidence of diabetes during the total follow - up . This was especially apparent when the success in achieving the goals was assessed at year 3, which probably reflects the importance of sustained lifestyle changes (13). The hazard ratios were 1.00, 0.87, 0.67, 0.70, and 0.23, for success scores from 0 to 45, respectively (p for trend <0.001). The effects of various components of intervention are interesting, and therefore some post hoc analyses related to this issue were completed . The independent effects of achieving the success score components at 3-year examination were assessed by including each of the five lifestyle goal variables individually in a cox model (table 1). Univariate hazard ratios for diabetes incidence (95% ci) were 0.45 (0.310.64) for weight reduction from baseline, 0.65 (0.450.95) for intake of fat, 0.59 (0.311.13) for intake of saturated fat, 0.69 (0.490.96) for intake of fiber, and 0.62 (0.460.84) for physical activity, comparing those who did or did not achieve the respective goal . When all the five success score components were simultaneously included in the cox model, the multivariate - adjusted hazard ratios for diabetes (95% ci) were 0.43 (0.300.61) for weight reduction, 0.80 (0.481.34) for intake of fat, 0.55 (0.261.16) for intake of saturated fat, 0.97 (0.631.51) for intake of fiber, and 0.80 (0.571.12) for physical activity . Furthermore, weight change was significantly associated with the achievement of each of the other four lifestyle goals, and consequently, success score was strongly and inversely correlated with weight reduction (27). Multivariate logistic regression model to predict diabetes during a 10-year follow - up correspondingly, the reduction in body weight was reported to be the main determinant of risk reduction in the u.s . After adjustment for other components of the intervention, there was a 16% reduction in diabetes risk per 1 kg weight lost during the first year of the intervention . Furthermore, lower percent of calories from fat and increased physical activity predicted weight loss, and increased physical activity was important to help sustain weight loss . Achieving the physical activity goal of 150 min / week reduced diabetes risk, especially among those participants who did not achieve the weight reduction goal of 7%, with risk reduction of 44% compared with those who achieved neither the weight reduction nor the physical activity goal . These findings suggest that dietary composition and physical activity are important in diabetes prevention, but their effect on diabetes risk is primarily mediated through resulting weight reduction . Nevertheless, because of multicolinearity it should also be noted that in the indian diabetes prevention program (15) and chinese prevention study (14), the participants were relatively lean, and there was no large change in body weight, but despite that, a remarkable reduction in diabetes risk was apparent . Thus, in these studies, components of the intervention other than weight control were responsible for the beneficial effects on diabetes risk . With compelling evidence that type 2 diabetes can be prevented or delayed, strategies to implement the primary prevention of type 2 diabetes both in high - risk subjects as well as at the population level are urgently needed . While type 2 diabetes prevention trials rigorously defined populations by explicitly characterizing their glycemic status, these studies did not include all groups at risk for developing type 2 diabetes . Methods that can also define other groups at high risk for developing type 2 diabetes have been recently developed and are increasingly used in several countries (28). The recent analysis of the dps has also shown that such people will significantly benefit from lifestyle interventions (27). A prospective study based on the data from the u.k . Estimated the association between the achievement of the five lifestyle goals used in the dps and the type 2 diabetes risk developing diabetes during a 4.6-year follow - up (29). The incidence of type 2 diabetes was inversely related to the number of goals achieved (p <0.001). None of the participants who met all five of the goals (0.8% of the total population) developed diabetes, whereas the risk of diabetes was highest in those who did not meet any of these goals . If the entire population were able to meet one more goal, the total incidence of diabetes is predicted to decrease by 20% . This finding suggests that health promotion interventions that result in an increase in healthy lifestyle in the general population might significantly reduce the growing burden of type 2 diabetes . Groups that will be the targets for prevention efforts can be identified through several reasonably effective strategies . However, there is no universal well - tested method that will identify all at high risk for developing type 2 diabetes, and there may be some variation in the optimal strategies for different populations and regions around the world . It is also important to realize that the identification of people having a high risk of type 2 diabetes or asymptomatic type 2 diabetes is not identical with the diagnosis of type 2 diabetes . In practice, we can identify people at high risk with simple and cost - efficient tools . The main question, however, is how to implement an efficient preventive strategy in individuals identified to be at high risk, i.e., how to translate the results of the recent successful type 2 diabetes prevention trials to a real - life setting (30). Much attention has been put on the biochemical methods for the assessment of glycemia in the early diagnosis of type 2 diabetes, but much less on the coverage of the detection of asymptomatic type 2 diabetes . The evidence is compelling that without applying an oral glucose tolerance test or an assessment of postprandial glucose, a large proportion of early cases of type 2 diabetes will remain unrecognized (31). The international diabetes federation consensus group recently prepared a document on the prevention of type 2 diabetes (32). This shows that the international diabetes community is now ready to accept the principle that the primary prevention of type 2 diabetes must be considered as an essential part of public health policy for diabetes . The american diabetes association consensus development conference in 2006 outlined principles regarding impaired fasting glucose (ifg) and igt and interventions to be applied among such individuals (26). The american diabetes association consensus group also recommended lifestyle intervention initially for people with ifg or igt (weight control and physical activity) but does not mention diet at all . If both ifg and igt are present as well as additional risk factors (and most of such people have additional risk factors), then a combination of lifestyle intervention and metformin is recommended . However, the evidence is not there to show that the combination of lifestyle and metformin is effective; on the contrary, the results from the indian diabetes prevention program suggest that there is no additional benefit from metformin over and above lifestyle intervention (15). It is not clear how much antidiabetic drugs can help in preventing progression from ifg or igt to overt diabetes and what is their overall costs and risk / benefit ratio in the long term . It is evident that long - term effects of lifestyle interventions are highly beneficial and that long - term costs are very low (13,26). (33) in their analysis stress that the real answer to reductions in incidence and prevalence of diabetes is in social policy, not in medical care.

The function of sensory receptors (nrec) in the movement control, muscle coordination and perception of the space position of temporo - mandibular joint (tmj) is fundamental although the presence of nrec in the tmj is still debated: some authors have reported on the lack of nervous fibers in the articular disk 1,2, while florid innervation of tmj has been reported in several studies on animal models and in human 3 - 6 which suggested that 6 the concentration of sensory receptors within tmj is higher in the areas supporting higher strong tensions during articular movements (chewing, biting, speaking). In discordance, other authors disclosed the presence of mechanical nrec in the articular disk of human tmj 7,8 also distinguishing receptors in capsulated and uncapsulated on the bases of morphological features . The aim of this study is to ascertain the presence and the distribution of nrec in human tmj by using of immunohistochemical investigations in healthy and pathological tmj such as arthritis and arthrosis . The study was approved by the bioethics committee of the department of odontology and surgery of university of bari . 10 samples of capsular and pericapsular soft tissues with the disk were obtained from healthy patients (six men and four women with a mean age of 39 years) who suffered surgery of tmj because of accidental trauma of the temporo - mandibular region; the remaining 7 cases (four men and three women with a mean age of 57 years) were patients surgically treated for severe degenerative lesions of tmj (chronic arthritis and arthrosis). All specimens were immediately fixed in neutral buffered formalin and embedded in paraffin; 5 micron thick sections were cut and stained with haematoxylin - eosin, pas, gomori's reticulin and azan - mallory trichrome; consecutive sections were used for the immunohistochemical detection of the antigens listed in table 1 . All the antibodies used are commercially avalaible from dako italia spa, milan, italy (glial fibrillary acidic protein = gfap; myelin basic protein = mbp; neurofilaments = nf; neuron specific enolase = nse; synaptophysin; s-100 protein = s-100) and from becton dickinson, burlingame, usa (leu-7). In all cases the immunohistochemical alkaline phosphatase - anti - alkaline phosphatase (apaap) method was performed 9 while some sections for anti - neurofilaments antibodies were treated for 10 minutes with 1% saponin in phosphate buffered saline (pbs), ph 7.2, before the application of primary antibodies . With histochemical techniques and h&e stain, nrec were not easily detectable (figures 1 - 8); however, these were used to identify the corresponding tissue in immunostained slides . In all specimens, such types of nrec have been detected by immmunohistochemistry (table 2): a) globular receptors with a thin capsule closely resembling ruffini's ones; they exhibited strong immunoreactivity in their core for s-100, nse and leu-7 and were abundant in the superficial peri - articular muscles and in the peri - articular fibrous capsule; b) elongated, onion - like receptors with a thick capsule mimicking pacini's receptors, positive for mbp, s-100, nse and leu-7, particularly abundant in deep muscle fibres and in peri - articular dense fibrous tissues; c) fusiform capsulated receptors, morphologically similar to golgi's receptors, located within peri - articular fibrous tissues and ligament and fibromuscolar resections, strongly positive for s-100, mbp and leu-7 and weaker gfap reactivity . In all these types of nrec (a+b+c) and especially in the core, punctate reactivity for synaptophysin and for neurofilaments was also evident; d) free and thin nervous endings in high density within subsynovial connective tissues, in intra- and periarticular fibrous tissues and along the perimisial, endomisial sarcolemma of striated muscle fibres were detected, showing immunoreactive for neurofilaments, nse, synaptophysin and to s-100 antibodies . In the articular fibrous cartilage (articular disk) any previously described nrec was identified; only s-100 protein seemed to react with chondrocytes both in normal and diseased tissues . Furthermore, chondrocytes of healthy individuals appeared round - shaped, with distinct cell borders and central nuclei with an evident s-100 reactivity both in nucleus and in cytoplasm . In diseased tmj, instead, chondrocytes showed a different morphoology, especially after s-100 immunostaining: they had an elongated cytoplasm with one or more thick dendritiform processes of variable length but with a strong reactivity only for s-100 protein . The number of dendritiform chondrocytes was higher in specimens of diseased patients than in healthy patients and seems undergo a reactive reparative proliferation of discal and peridiscal tissues . Few studies are reported in literature on the precise identification and distribution of nrec in articular and peri - articular tissues of tmj . In the past, some authors 3 - 6 identified ruffini's - like, pacini's like and golgi's like receptors both in articular and periarticular tissues, using conventional or histochemical methods usually performed to identify nerve fibres and receptors 7,8 . By using immunofluorescent techniques, other authors 3 demonstrated the presence of nervous fibers in the periarticular fibrous tissues, which seemed to run along the blood vessels reaching the fibrous cartilage of tmj and ending to the inside . Our study confirmed the results of other preceding reports on the presence of several different types of nrec in periarticular soft tissues of tmj, allowing additionally a precise immunohistochemical identification of ruffini's - like, pacini's- like and golgi's - like receptors in skeletal muscles and tendons, in periartcular dense fibrous connective tissues and in subsynovial tissues . In fact, nrec appeared nse, s-100 and mbp immunoreactive showing gfap and and leu-7 immunoreactivity to lower degrees . Free nervous endings, immunohistochemically positive for neurofilaments, nse, and s100 protein, have been detected only in periarticular soft tissues (higher density in muscles and in the vascular venous plexus in the posterior part of discal ligaments and in the trilaminar zone) and not in cartilagineous disk . The latter, besides, appeared constituted by s100 immunoreactive chondrocytes both in healthy individuals and in patients with chronic degenerative tmj lesions . In pathological patients with severe disk damage number and morphology of chondrocytes were severely different in comparison to normal tissues 11; chondrocytes, in fact, were more numerous with a rough and thick elongated cytoplasmic processes conferring a dendritic - like appearance . In some instances, dendritic processes were extremely long and consequently their cytoplasm of origin could not be detected in a single section but only on consecutive sections of the same specimen . In no instances this cellular component and their prolongments in cartilagineous disks showed neurofilaments, nse or synaptophysin immunoreactivity . Morphological changes of chondrocytes in patients with chronic arthritic disease of tmj we observed, conferring them the appearance of neural cells and their axons, have been previously described in literature 10 . This misleading feature is further emphasized by the occurence of s-100 immunoreactivity in dendritic chondrocytes, as well as in normal chondrocytes . Nevertheless, s-100 positive cells in the articular cartilage of tmj bear rare, thick and coarse cytoplasmic processes in which no one of the antigens commonly found in peripheral nerve fibres (e.g. Leu-7, mbp, neurofilaments, nse and synaptophysin), except for s-100, could be detected . In contrast, peripheral nerve fibres usually exhibit long, thin and varicose cytoplamic prolongments in which a variable combination of the above mentioned antigens is usually detectable in conjunction with s-100 reactivity . The results of our study indicate that free nervous endings described by other authors 3 - 8 are definitely proven to be nrec, and it appears likely to stress that, especially in pathological conditions, chondrocytes and their prolongments might morphologically resemble nrec in the articular disk, although their immunophenotype is rather different with the lack of expression of typical neural antigens . Although immunochemistry can be easily used to study distribution and location of nrec in articular tissues, we also suggest that ultrastuctural and immuno - ultrastructural studies should be performed in order to definitely assess if chondrocytes is the exclusive cell type of articular cartilage or if also nrec could be present in tmj.

Areview on the history of mental disorders shows their wide prevalence among human beings always . A glance on statistics reveals that mental disorders and abnormalities are affecting the humans at an increasing pace . The world health organization (who) in its report revealed that the incidence of mental disorders is 10% among the young adults . Noorbala et al ., in a study on mental health among individuals> 15 years of age (n = 19,370) who were randomly selected, reported that 34.2% of the subjects had different types of mental disorders . On the other hand, university students as a major social group and human resource are the future investment in each society and their mental health is of great importance . The above - mentioned statistics show a high prevalence of the disorders among university students . On the one hand, the university entrance process influences students mental health through educational stressors, immigration to major cities, being far from the family, facing financial problems, etc . On the other hand, if these students face an additional stress due to the stressful nature of their major, their mental health should be considered more . Among the stressful educational courses are nursing and midwifery . Based on a study on the association between mental health and educational stressors in nursing students of kerman nursing school in 2011, an increase in the number of students referrals to counseling centers shows their social and cultural growth on one hand, as asking help is believed to be a sign for mental growth, but on the other hand, it reveals the disturbing factors decreasing students academic performance . In a study conducted in private universities of malaysia, therefore, experts of education, psychologists, sociologists, and educational policy makers should seek an appropriate pattern to facilitate students overall growth and their mental health improvement . Regard, realizing the need of humans, scientists tried to explore and reconstruct more humanistic methods consistent with human beings psychological characteristics to reach their mental health . Therefore, in the recent past, some psychologists have turned to new educational and treatment methods, namely, neurolinguistic programming (nlp). In addition, a wide spectrum of human behaviors are mediated and regulated by human language . The importance of nlp lies in the fact that this programming is a collection of skills based on psychological characteristics of the human beings through which the individuals obtain the ability to use their personal capabilities as much as possible . In an article titled neurolinguistic methodology in controlling examination anxiety reported that anxiety is one of the destructive factors of students mental health in every level and can reduce educational function . They introduced nlp as one of the efficient methods to control pre - exam anxiety . As mentioned earlier, students of medical group, including nursing and midwifery, tolerate high stress due to the stressful nature of their course, not only during their education but also at their working environment, and need decision making in critical situations . With regard to their various employment opportunities and working conditions in hospitals, which can affect their mental health, the researcher conducted a study with an aim to define and compare the efficacy of nlp training on mental health in nursing and midwifery students in islamic azad university, tehran medical branch in 2011 - 2012 . The sample size was calculated as 30 subjects in each group by alpha = 0.05, z = 1.96, and delta = 0.80 . Due to subjects dropping out of the study in the midwifery group, finally 52 subjects entered the study in both groups . Both groups were homogenous concerning age range, sex, education level, and the faculty and the university . Group comparison was conducted with regard to adequacy of sample size(based on sample size formula). The first section was on personal characteristics including age, sex, course of study, year of entering university, marital status, place of birth, residence, head of household, parents education and family monthly income, history of psychotropic drug, stimulants, and narcotics consumption, and history of a disastrous experience in the past 6 months . The second section consisted of 28-item goldberg general health questionnaire, which had four subscales of physical signs, anxiety, social dysfunction, and depression . The total score of general health was obtained for the subjects by adding up the scores of these four sub - scales . Questions 1 - 7 were connected with physical signs, 814 with anxiety, 15 - 21 with social function, and 2128 were connected with depression . Each question had four options, with scores ranging from 0 to 3 (never = 0, normal = 1, almost more than normal = 2, and actually more than normal = 3). The total scores ranged 0 - 84, of which scores> 23 showed low general health . Reported the general validity of this test as 88% and that of its subscales to be between 50% and 81% . They also reported the reliability of its subscales as: physical signs = 0.78, anxiety = 0.86, social dysfunction = 0.77, and depression = 0.88 . Then, the educational program in the form of a 1-month program containing five 120-min educational sessions was held . The sessions were in lecture form and as questions and answers accompanied with slide show and short films . An education booklet and a cd were prepared from valid references prior to each educational session and distributed among the attendants . During the sessions, some skills of nlp were taught: (1) defining a goal, (2) time management, and (3) self - assertion . Then, 1 month after the last educational session, subjects mental health was measured with the same questionnaire . Data were analyzed by descriptive (absolute and relative distribution table, mean and sd) and inferential (paired t - test) statistical tests . Ethical considerations of the present study included obtaining consent from the subjects and giving them explanations about the goal of the study . They were also assured of confidentiality of their data and the mere use of the data for analysis . Some limitations, which were out of the researchers control, were personal differences (different levels of motivation, comprehension, interest of the subject, and iq) and social and cultural differences, which could have affected the subjects level of learning . Subjects honesty in responses and the relevancy of their answers were out of control of the researchers . Lack of a control group due to less number of volunteers and educational classes interference with students own classes were the other limitations of this study . About 57.7% of the subjects were nursing students and 42.35% were midwifery students, 80.8% were single and 19.2% were married, 78.8% were born in tehran and 21.2% in other cities, 19.2% consumed psychotropic medications and 13.5% had a history of referring to a psychiatrist, and 30.8% smoked in their leisure time . Mean scores of nursing students mental health dimensions were 56.23 (14.7) before nlp education and 17.36 (9.44) after nlp education . Score of mental health showed a significant reduction of 39 scores after education compared to before education . In other words, nlp education had a significant effect on the reduction of mental health scores (p <0.001, t = 18.42) [table 1]. With regard to subscales, the mean score of physical signs dimension was 14.30 (4.78) before education and 4.76 (3.63) after education (p <0.001). The mean score of anxiety was 15.10 (4.22) before education and 4.76 (3.63) after education (p <0.001). The mean score of depression was 10.73 (5.38) before education and 1.01 (2.27) after education (p <0.001). The mean score of social function was 16.10 (2.52) before and 8.53 (3.63) after intervention (p <0.001). The mean score of depression was 10.37 (5.38) before and 1.01 (2.27) after intervention (p <0.001). A reduction of mean score observed in all dimensions after education shows the positive effect of nlp education . The mean score of mental health among midwifery students was 53.72 (15.97) before education and 23.63 (12.06) after education, which is significantly less than pre - test values, showing that nlp was effective on the subjects mental health (p <0.001) [table 2]. With regard to mental health dimensions, the mean score of physical signs was 11.36 (5.10) before education and 5.54 (4.30) after education (p <0.001). The mean score of anxiety was 14.54 (6.32) before education and 5.54 (4.35) after education (p <0.001). The mean score of social function was 16.36 (3.23) before education and 10.01 (3.54) after education (p <0.001). The mean score of depression was 11.45 (5.56) before education and 2.54 (3.71) after education (p <0.001). Determination and comparison of nursing students health status before and after education determination and comparison of midwifery students health status before and after education in the nursing group, the mean score of mental health was 56.23 (14.7) before education and 17.36 (9.44) after education, while in the midwifery group, it was 53.72 (15.97) before and 23.63 (12.06) after education . There was no significant difference in the mean scores of general health before education concerning the course of study (p = 0.561), but the difference was significant after education in nursing and midwifery subjects such that nursing students mental health was more influenced by nlp education compared to that of midwifery students (p = 0.041). In the nursing group, the mean score of physical signs was 14.30 (4.78) before education and 4.76 (3.63) after education and in the midwifery group, it was 11.36 (5.10) before education and 5.54 (4.30) after education, with no significant difference in the mean scores of physical signs concerning the course of study before and after nlp education (p = 0.584). In the nursing group, the mean score of anxiety was 15.10 (4.22) before education and 3.06 (3.01) after education and in the midwifery group, it was 14.54 (6.32) before and 5.54 (4.35) after education, showing a significant difference concerning the course of study (p = 0.019) such that anxiety in the nursing group was significantly higher than in the midwifery group before education . After nlp education, the anxiety in nursing group was less than in midwifery group, revealing the effect of nlp education on the nursing group . In the nursing group, the mean score of social function was 16.10 (2.52) before education and 8.53 (3.63) after education and in the midwifery group, it was 16.36 (3.23) before education and 10.01 (3.54) after education, revealing no significant difference in the mean scores of social function with regard to the course of study before and after education (p = 0.153). In the nursing group, the mean score of depression was 10.73 (5.38) before education and 1.01 (2.27) after education and in the midwifery group, it was 11.45 (5.56) before education and 2.54 (3.17) after education, showing no significant difference (p = 0.153). The mean scores of mental health pre - test were significantly different in the groups with respect to the variables of place of birth, interest in the selected course, interest in the present course, mental disease, consumption of psychotropic medications, referring to a psychiatrist, existence of a disastrous event 6 months prior to study, and smoking and alcohol consumption (p = 0.05). Mental health was lower among the students of tehran compared to that in students of other cities . It was also lower among the students who were not interested in their course of study compared to those who were . The students with a history of mental disease and referral to a psychiatrist had lower mental health compared to those with no such history . Mental health among the students experiencing a disastrous event in the past 6 months, or consuming psychotropic medication, cigarettes, or alcohol was lower than that of others . Mean scores of students mental health showed a significant difference with regard to smoking a water pipe (p = 0.005). Mental health was lower among the students who always smoked cigarette and a water pipe compared to those who did not . Comparison of pre - test post - test mean scores of students mental health showed no significant difference in the variables associated with family (p> 0.05). In the nursing group, the mean score of mental health was 56.23 (14.7) before education and 17.36 (9.44) after education, while in the midwifery group, it was 53.72 (15.97) before and 23.63 (12.06) after education . There was no significant difference in the mean scores of general health before education concerning the course of study (p = 0.561), but the difference was significant after education in nursing and midwifery subjects such that nursing students mental health was more influenced by nlp education compared to that of midwifery students (p = 0.041). In the nursing group, the mean score of physical signs was 14.30 (4.78) before education and 4.76 (3.63) after education and in the midwifery group, it was 11.36 (5.10) before education and 5.54 (4.30) after education, with no significant difference in the mean scores of physical signs concerning the course of study before and after nlp education (p = 0.584). In the nursing group, the mean score of anxiety was 15.10 (4.22) before education and 3.06 (3.01) after education and in the midwifery group, it was 14.54 (6.32) before and 5.54 (4.35) after education, showing a significant difference concerning the course of study (p = 0.019) such that anxiety in the nursing group was significantly higher than in the midwifery group before education . After nlp education, the anxiety in nursing group was less than in midwifery group, revealing the effect of nlp education on the nursing group . In the nursing group, the mean score of social function was 16.10 (2.52) before education and 8.53 (3.63) after education and in the midwifery group, it was 16.36 (3.23) before education and 10.01 (3.54) after education, revealing no significant difference in the mean scores of social function with regard to the course of study before and after education (p = 0.153). In the nursing group, the mean score of depression was 10.73 (5.38) before education and 1.01 (2.27) after education and in the midwifery group, it was 11.45 (5.56) before education and 2.54 (3.17) after education, showing no significant difference (p = 0.153). The mean scores of mental health pre - test were significantly different in the groups with respect to the variables of place of birth, interest in the selected course, interest in the present course, mental disease, consumption of psychotropic medications, referring to a psychiatrist, existence of a disastrous event 6 months prior to study, and smoking and alcohol consumption (p = 0.05). Mental health was lower among the students of tehran compared to that in students of other cities . It was also lower among the students who were not interested in their course of study compared to those who were . The students with a history of mental disease and referral to a psychiatrist had lower mental health compared to those with no such history . Mental health among the students experiencing a disastrous event in the past 6 months, or consuming psychotropic medication, cigarettes, or alcohol was lower than that of others . Mean scores of students mental health showed a significant difference with regard to smoking a water pipe (p = 0.005). Mental health was lower among the students who always smoked cigarette and a water pipe compared to those who did not . Comparison of pre - test post - test mean scores of students mental health showed no significant difference in the variables associated with family (p> 0.05). The results obtained showed that nlp education was effective on the mental health of nursing and midwifery students (p <0.001). These results are consistent with the results of sahebalzamani et al ., who conducted a study on the effect of life skills training on general health in students and showed that life skills training is effective on nursing and midwifery students mental health (p <0.01, t = 11.2). Our results are consistent with the result of zamini et al . Who studied the effect of nlp education on self - efficiency and problem solving among students . Our results showed that nlp education is notably effective on students mental health, indicating that students general health can be promoted through nlp education . The results showed that the mean scores of mental health were 56.23 (14.7) and 53.72 (15.97) in nursing and midwifery groups, respectively, before education, showing no significant difference with regard to the course of study (p = 0.561). But they were 17.36 (9.44) and 23.63 (12.06) in nursing and midwifery groups, respectively, after education, showing a significant difference with regard to the course of study (p = 0.041). It can be concluded that mental health was higher in the nursing group after they were given nlp education, compared to the midwifery group . In other words, nlp education had a notable effect on nursing students mental health promotion . Results of this study concerning mental health dimensions showed that anxiety was more influenced by education in nursing students, which needs further studies investigating more dimensions in this regard . The obtained results are consistent with of the result of sahebalzamani et al . Reporting the effect of expression skill education on self - esteem and decisiveness of high - school girls (p = 0.000). With regard to the increasing changes and sophistication of society and development of social communications, preparation of individuals, especially the young generation, for facing difficult situations is an essential issue . Since iran has a young population, and unfortunately, corruption and social assaults have increased in recent years, the policy makers of education and training system and the related authorities should essentially have plans to lower the corruption and assault and to increase job opportunities in the society and create jobs for students . Our results showed that nlp education can promote individuals mental health level . By providing nlp education skills including setting a goal, time management, and the skill of self - expression in the form of nlp nlp education, as public education, can increase the level of knowledge and have a positive effect on the various dimensions of human life . As students are the future investment of the society, their mental health is of great importance . A family is the first place for children to experience a common life and so ciability . Therefore, based on the findings, it is suggested to consider nlp education along with other educational courses, not only to promote individuals general health but also to lower depression, anxiety, social function reduction, and physical problems, which will prevent mental and physical disorders.

Polycystic ovary syndrome (pcos) which was first reported in 1935 is known as one of the most common endocrine hormones disorders in the women of the reproductive age afflicting as many as% 10 of them . The name of this syndrome has been derived from the appearance of the ovaries of the afflicted women (an ovary of enlarged size and full of cysts). These cysts are in the form of follicles filled with liquid sacs full of developed ovum . Due to the lack of a set of standard and unified diagnostic criteria, the rate of prevalence of this syndrome reported in the different studies is varied . Some studies which used sonography as their diagnostic criteria found that the prevalence of pcos ranged between 21% and 22% respectively . On the contrary, in some other studies using oligomenerrhea and hyperandrogenemia manifestations as the pcos's diagnostic criteria, this syndrome accounts for 30 - 40% of the common causes of infertility resulting from ovary dysfunctions . In addition, pcos is considered as the most leading cause of ovary disorders . As recent studies indicate, a person may have pcos without showing one or some of the above - mentioned symptoms, some other symptoms and signs which can be enumerated for pcos are as follows: increased androgenic hormones, metabolic syndrome (insulin resistance), fat - related disorders, type 2 diabetes, cardiovascular disease, endometrial cancer and blood pressure . The patients with pcos also suffer from anxiety and depression some symptoms which are believed to be mostly a result of disorders occurred in the reproduction cycle which is naturally completed by releasing an ovum every month . The exact cause of pcos is not yet completely known, however, some factors such as insulin resistance / hyperinsulinaemeia, obesity, heredity and genetics factors, environmental factors such as exposure to high levels of masculinizing hormones, embryonic life cycle - related factors, hyperandrogenemia, gonadotropins secreting and functioning, hyperprolactinemia, hypothyroidism or thyroid - related dysfunctions, diet all may play a part in developing pcos . According to previous studies, the eating habits are not different between patients with pcos and healthy persons but some foods seem to affect hormone parameters . In the year 2008, one case - control study was carried out in al - zahra hospital of the city of tabriz, iran in which 60 women were studied, the results showed that the intake amounts of ca, mg, d vitamin, dairy, fruits and nuts and seeds were remarkably low among the women with pcos . The frequency of their milk and dairy intakes products was also lower compared with the healthy women (p <0.05). Another study by billaudel et al . Revealed that d vitamin and calcium play a critical role in insulin secretion and insulin - resistance in human and animal models . Another research on 103 women with pcos and 103 healthy women provided some evidence on the decreased amount of adiponectine, calcium, d vitamin in the patients suffering from pcos with having a higher thyroglobulin . There was also a significant correlation between adioponectrine and calcium concentrations (oh - d 25). In one prospective study on 3157 young women aged 18 - 30, pereira et al . Have reported in 2002 that the amount of dairy intake and insulin - resistance were indirectly related . A study by pereira et al . Found an inverse relationship between consuming more than 4 units of dairy products per day and metabolic syndrome in the females . As indicated in previous research, diet and dairy intake which itself is a source of calcium not only can trigger pcos condition, but also may directly affect obesity and insulin - resistance, two factors which themselves act as the causes of pcos . Numerous studies have demonstrated the association between the diet and its components and risk factors developing various diseases . However, previous studies did not address the relationship between nutrition choices and type of diet chosen by the patients . The adverse health effects of pcos such as infertility, diabetes, cardiovascular diseases and the growth in its prevalence, can impose huge financial and health costs on the society and can affect affecting many families . Therefore, however, the number of studies which have addressed the relationship between dairy intake and pcos is limited . Hence, the present study intends to explore the relationship between the amount of dairy intake and pcos . As a descriptive cross - sectional study, the present research tried to find the relationship between the amount of dairy consumption and pcos condition in 400 women who referred to shahid beheshti hospital clinic in 2013 . As far as exclusion and inclusion criteria of the study is concerned, it is noteworthy that the criteria for the inclusion in the study were willingness to participation, lack of precocious puberty, not having uterus cancer and not being pregnant . On the contrary, the subjects were excluded from the study if they were unwilling to participation, did not respond to more than 30 food items included in the food frequency questionnaire (ffq), diagnosed with typical (chronic) diseases having to follow a specific diet . At the start of the study, the data related to the food intakes were checked by the researcher using ffq, (in the ffqs the person were asked how often they consumed certain foods from a given l of foods) the validity and reliability of which were previously confirmed . The data on the history of developing ovary dysfunctions (any endocrine disorders related to reproductive system), genetics background (having first related family members with ovarian disease), the age of the first menstruation, the history of thyroid diseases, the patients medication history (any kind of medicine used or any surgery), the period of developing pcos and physical activity were collected by a nutritious expert using a self - developed questionnaire . To do so, the weight (kg) of the participants was measured with clothes but without shoes . The height of the participants was measured in a normal condition without wearing shoes using a strip tape . The body mass index (bmi) was calculated by dividing the weight (kg) value by the square root of height (m) value . The pcos condition was identified through sonography diagnostic assessment with checking patients clinical manifestations performed by a trained authority . Data analysis was performed by descriptive analysis and logistic regression tests using spss software version 15 . For age, age at menarch and bmi t - test was applied . Mann - whitney t - test was applied for kcal while for other variables chi - square test was applied [table 1]. As shown in table 2 for all variables mann - whitney test were used . The study's confidence coefficient was considered at 95% and the level of strength of the study at 80% . Furthermore, the ratio of pcos - afflicted patients and the sampling error were assumed to be 50% and 0.07%, respectively . Frequency (%) and central tendency (spread) of some health condition characteristics of participants frequency (%) and central tendency (spread) of food intake characteristics of sample population study as a descriptive cross - sectional study, the present research tried to find the relationship between the amount of dairy consumption and pcos condition in 400 women who referred to shahid beheshti hospital clinic in 2013 . As far as exclusion and inclusion criteria of the study is concerned, it is noteworthy that the criteria for the inclusion in the study were willingness to participation, lack of precocious puberty, not having uterus cancer and not being pregnant . On the contrary, the subjects were excluded from the study if they were unwilling to participation, did not respond to more than 30 food items included in the food frequency questionnaire (ffq), diagnosed with typical (chronic) diseases having to follow a specific diet . At the start of the study, a written information - consent form was signed by the participants . The data related to the food intakes were checked by the researcher using ffq, (in the ffqs the person were asked how often they consumed certain foods from a given l of foods) the validity and reliability of which were previously confirmed . The data on the history of developing ovary dysfunctions (any endocrine disorders related to reproductive system), genetics background (having first related family members with ovarian disease), the age of the first menstruation, the history of thyroid diseases, the patients medication history (any kind of medicine used or any surgery), the period of developing pcos and physical activity were collected by a nutritious expert using a self - developed questionnaire . The weight and height of the participants were also measured . To do so, the weight (kg) of the participants was measured with clothes but without shoes . The height of the participants was measured in a normal condition without wearing shoes using a strip tape . The body mass index (bmi) was calculated by dividing the weight (kg) value by the square root of height (m) value . The pcos condition was identified through sonography diagnostic assessment with checking patients clinical manifestations performed by a trained authority . Data analysis was performed by descriptive analysis and logistic regression tests using spss software version 15 . For age, age at menarch and bmi t - test was applied . Mann - whitney t - test was applied for kcal while for other variables chi - square test was applied [table 1]. As shown in table 2 for all variables mann - whitney test were used . The study's confidence coefficient was considered at 95% and the level of strength of the study at 80% . Furthermore, the ratio of pcos - afflicted patients and the sampling error were assumed to be 50% and 0.07%, respectively . Frequency (%) and central tendency (spread) of some health condition characteristics of participants frequency (%) and central tendency (spread) of food intake characteristics of sample population study this study was carried out on 400 women of an mean standard deviation (sd) age of 29.5 4.8 years old and an mean sd bmi of 24.1 4.8 admitted to the shahid beheshti hospital . The prevalence of pcos in the persons in the study was found to be 9.9% . Based on the primary analyses on the data (descriptive analysis), there was relationship between the history of ovarian diseases, genetics and the use of medications and pcos . No relationship was observed between pcos and other variables [table 1]. As for the history of ovarian diseases, there was statistically significant difference between the patients with pcos and healthy people (p <0.001). As for the family history, a similar result was obtained so that 22.6% of the patients with pcos were revealed having a family history of pcos . On the contrary, there was no significant relationship between physical activity and pcos (p = 0.86). A significant relationship was also observed between the use of medications and pcos (p 0.001). The pcos - afflicted patients and healthy persons did not show any difference in terms of weight and bmi . The average age of the patients with pcos was 26.7 compared to 29.8 as the average age of the healthy persons . The two groups also differed in terms of the amount of calorie intake with 4247 kcal for the subjects with pcos and 6410 kcal for the healthy subjects . The average age of the first menstrual period was found to be 13.35 years among the healthy individuals and 13.02 years among the individuals afflicted with pcos [table 1]. The average sum of dairy intake was 581.5 and 611 g for the healthy persons and patients with pcos, respectively [table 2]. The results from using logistic regression revealed that there was no association between genetics and pcos (p = 0.13) [table 3]. On the contrary (p <0.001 odds ratio [or] = 0.2 confidence interval [ci] (95%) = 0.082 - 0.490), age (p <0.001, or = 0.85 ci (95%) = 0.787 - 0.934) and the use of medication as three confounding variables (p = 0.001 or = 0.17 ci (95%) = 0.064 - 0.477) and pcos [table 3]. Based on the results, bmi and pcos were not significantly related but their relationship may be remarkable (p = 0.068). Every one - unit increase in the bmi was accompanied by an increase in the risk of affliction with pcos . Although like bmi, this relationship was not significant but remarkable (p = 0.088). After modifying the effects of confounding variables, a direct significant relationship was found between the intake of milk and pcos (p = 0.028) so that every 1-unit increase in the milk intake led to a% 1-increase in the risk factors [table 3]. In the present study we evaluated the effects of dairy product on pcos . According to the results of this study, no significant relationship was observed between each component of dairy foods group and other variables with pcos . However, after modifying the effects of confounding variables, it was found that the milk intake and pcos were directly related . Very few studies have been conducted on exploring the direct effect which dairy foods may have on pcos . The results of empirical research on the effect of dairy foods on the weight which itself is believed to be one of the effective factors of infliction with pcos are inconsistent without producing a rigid and strong outcome . The results of available clinical evaluations especially those who address the effects of a diet rich in dairy foods on the weight do not yield a fixed outcome . The results of some previous studies are in line with our results . According to adebamowo et al ., there is a relationship between slim / low - fat or free - fat milk and increased frequency of acne, a component of the pcos . Two intervention studies (thompson et al . And harvey - berino, et al .) Provided no evidence on an additional weight loss when choosing a diet rich in dairy foods compared with a low - dairy diet . The results of these studies were similar to those of one earlier research by zemel et al . (2004) with a study period of 12 weeks . However, the periods of these two recent studies in which no association was observed between the variables in question was 48 weeks and 52 weeks, respectively . The findings of a 3-year prospective study on a large number of children and young adults revealed that the subjects with a higher intake of milk had a higher bmi . Although this reported increase was very trivial per year, adjusting for a number of body weight factors, it was statistically significant . Chavarro et al . In their prospective cohort study found that high intake of low - fat dairy products may lead to an increase in the women's risk of ovulation - related infertility, while incorporating more high - fat dairy foods may decrease its risk . (1994) reported a positive association between intakes of milk and age - related decreased fertility rates in 31 nations . To put it differently, consuming milk gunther et al . In their study divided 155 women of normal weight into three sub - categories as per their amount of dairy foods intake within 1 year, while their diets during intervention were the same in terms of caloric value . Although the results of the study did not support the hypothesis related to the dairy foods intake and weight loss, following 51 of the healthy women participating in the study for a 6-month period after the study revealed that compared to those with a low intake of dairy foods, those who had a diet of high dairy intake had a lower fat accumulation . Results of some previous studies are not in line with our findings . In two studies by zemel a considerable decrease was reported for bmi and waist circumference with a notable decrease in the plasma insulin . In one case - control study, dixon, et al . Argued that there is an intricate association between obesity and dairy products intake concluding that for the children of higher age, there was an inverse association between the intake of dairy foods and obesity . By analyzing a number of databases, a group of cross - sectional studies on the adults and children almost demonstrated the inverse relationship between the intake of dairy foods and initial obesity . The samples used in these studies were mostly selected from among post - menopausal middle - aged women . Carruth and skinner in their research on a group of preschool children concluded that the number of servings estimated per day is obviously inversely related to the fat accumulation and positively related to the individuals slimness . In particular, each serving resulted in an additional weight loss up to 0.9 - 1.1 kg . As for consuming dairy foods and insulin resistance relationship, a cross - sectional study conducted on 496 samples of participants aged 20 - 68 aged in 2009 can be enumerated . This study tried to assess the amounts of consumed dairy foods using a food history questionnaire the validity of which had been checked . Based on the results, compared with other groups, the samples situated on the top quarter of the intake of dairy products had the lowest mean scores for insulin resistance markers . Although the observed differences were not statistically significant, eating whole - fat dairy products was inversely associated with homa - ir . In contrast, there was no relationship between low - fat dairy products and insulin resistance markers . A case - control study by keshavarzi demonstrated that the risk of infertility among the women who ate 3 or more glasses of milk per day was lower than those with no intake of milk as much as 70% . Most of these studies were done on obese subjects, children or on adolescents . On the whole, the findings of this study indicated that there was no statistically significant relationship between consuming milk, yogurt, sherbet / frozen yogurt, cheese and cream and pcos . Conversely, it became evident that pcos and the history of ovarian diseases (p <0.001), age (p <0.001) and the use of medication (p = 0.001) as three confounding variables were inversely related . Furthermore, although no statistically significant relationship was obtained between bmi and pcos, it was at the threshold of being significant (p = 0.068). Every one - unit increase in bmi is associated with a 10% increase in the risk of pcos . Of course, this relationship was no significant . Similar to bmi, this relationship was insignificant (being at the threshold of significance, p = 0.0880 . After modifying the effects of confounding variables, a statistically significant relationship was observed between milk intake and risk of pcos (p = 0.028) hence that every 1-unit increase in the amount of milk consumed caused a 1%-increase in the risk of pcos . There is no study addressing the relationship between the history of ovarian diseases and pcos . As for the relationship between the use of medication and pcos, the research suggest that since the medications used by the majority of the participants were metformin, anti - fertility as well as thyroid medications, they helped prevent developing pcos due to their effect on androgen secretion and luteinizing hormone / follicle - stimulating hormone ratio . Based on past research, the higher the age, makes the lower the possibility of pcos . Affecting ovulation however, it is noteworthy that yielding inconsistent results, few studies have addressed this issue . If the women with pcos or those who are suspicious of having it make some changes in their diets, for instance choosing low - fat dairy foods instead of high - fat ones, a positive relationship between these changes and anovulatory infertility would be expected . Foods factors relevant to the augmentation of the phenotype attributes of pcos may increase infertility risk due to disturbed ovulation . The current study revealed that the average intake of milk and pcos had significant relationship . To explain more, on the average the total intake of low- and free - fat milk was estimated to be 52 61 (gr) and 55 132 (gr) for the healthy and patient individuals, respectively . The average amount of low- and free - fat milk consumption was found to be 12.4 39.35 (gr) for the healthy persons and 76.55 (gr) for the pcos patients . Furthermore, the individuals with pcos condition, revealed to have a higher consumption of low- and free - fat milk . As per the results of the past studies, there is a positive relationship between low - fat milk and pcos . Low - fat dairy products may increase the level of insulin like growth factor i (igf - i). As some researchers have controversies the igf - i existing in the human ovarian cells may stimulate the cell activity - related changes observed in the pcos . It is not yet known whether the change in the level of igf - i caused by diet may contribute to the pcos's clinical manifestations or not . As the research suggest, the fatty acids of dairy products may have potential positive effects on the ovary function . In more details, the substance soluble in the fat available in the dairy products plays the essential role in creating such effects . Compared with low - fat dairy products, whole milk and fat - rich dairy products since estrogen can decrease the level of igf - i, their existence in high - fat dairy foods may explain the observed association . The frequent increased insulin sensitivity in the high - fat dairy consumers may improve their ovulation performance . Eating low - fat dairy foods has also been accompanied by an excess of androgens secretion known as one of components of pcos . One of the weak points of this study is its cross - sectional design which fails to identify the cause - effect relationships . In addition, hormone tests such as thyroid, prolactine and homa - ir tests were not taken from the subjects . In contrast, some strength points that can be mentioned for this study include: the large number of the samples, lack of similar study in iran directly addressing the relationship between dairy products intake and pcos, evaluating the foods intakes using a validated ffq, using sonography test known as the most reliable method for pcos diagnose . Based on the findings of the study, milk intake and prevalence of pcos may be related in some way . Due to the adverse health effects of this condition, its high prevalence in the society as well as the shortage of research on the effectiveness of diets on preventing and treating it, the need to more research is felt . In the future research, it is better to address laboratory practices such as thyroid, prolactine and sexual hormones, as well.

Milrinone, a phosphodiesterase iii inhibitor currently used to treat pediatric patients with cardiac diseases, increases the level of cyclic adenosine monophosphate in the myocardium and vascular smooth muscle . High levels of cyclic adenosine monophosphate enhance the contractility of the myocardium by increasing calcium influx and relaxing vascular smooth muscles,1) therefore increasing cardiac output and decreasing afterload . Milrinone also has a lusitropic property, which improves myocardial relaxation.2) unlike other inotropes such as dopamine, dobutamine, and epinephrine, milrinone is not associated with an increase in myocardial oxygen consumption.3) milrinone is widely used due to its inotropic, vasodilatory, and lusitropic properties . It is frequently prescribed after cardiac surgery due to its efficacy in preventing low cardiac output syndrome.4) milrinone is also commonly administered to pediatric patients with myocarditis as a bridge therapy for patients with heart failure who are waiting for cardiac transplantation.5)6) milrinone has been shown to improve heart contractility in patients with septic shock.7) milrinone is currently one of the most commonly used off - label cardiovascular medications in children.8) although a few reports showed the efficacy and safety of short - term milrinone treatment (for about 35 hours) in children with heart disease,9)10) the safety and efficacy of long - term use of milrinone in children is limited due to a lack of sufficient evidence - based studies in pediatric populations . Currently, milrinone is widely used for 3 days, which is usually based on extrapolation from studies in adults or clinical experience . Therefore, the objective of this study was to evaluate the safety and efficacy of the current pattern of milrinone administration for 3 days in pediatric patients . A retrospective analysis was conducted using data collected from patients who received milrinone treatment for 3 days at the seoul national university children's hospital from january 2005 to december 2012 . Patients were divided into two groups based on the indication for milrinone treatment: group a consisted of patients who received milrinone to prevent low cardiac output syndrome after cardiac surgeries whereas group b consisted of patients who received milrinone treatment due to acute heart failures from causes other than cardiac surgeries . Patients with the following conditions or those who underwent the following treatments were excluded from the study: 1) severe left ventricular outflow obstructive diseases such as aortic stenosis; 2) hypertrophic or restrictive cardiomyopathies; 3) milrinone administration immediately after cardiopulmonary resuscitation; 4) multi - organ failures; 5) simultaneous administration of 3 inotropes other than milrinone to increase blood pressure; 6) renal replacement therapies including peritoneal dialysis; 7) myocardial infarctions; 8) univentricular heart surgeries . However, patients receiving 3 inotropes were included if inotropes were added after an interval of> 2 hours consecutively . Patients' characteristics including age, sex, height, weight, and body surface area were recorded . The following parameters were analyzed to identify the clinical application of milrinone: 1) initial infusion rate; 2) maintenance continuous infusion rate; 3) total duration of milrinone therapy; 4) concomitantly infused inotropes . The operation and anesthesiology records of patients who had undergone cardiac surgeries were also studied to identify data related to anatomic diagnosis, type of surgery, time of cardiopulmonary bypass, and time of aortic cross - clamping . Systolic blood pressure, heart rate, and echocardiographic data (the left ventricular internal dimension at end - diastole, ejection fraction, and fractional shortening) obtained before or immediately after starting milrinone treatment were compared to the values prior to treatment cessation . In cases where appropriate echocardiographic data could not be obtained on the day milrinone treatment was discontinued, echocardiographic data obtained within a week of the treatment endpoint were used . The safety of milrinone was determined based on the occurrence of adverse events such as hypotension, arrhythmia, chest pain, headache, hypokalemia, and thrombocytopenia . Hypotension was defined as a sudden decrease in blood pressure within two hours after starting milrinone that necessitated the addition of inotropes or cessation of milrinone . Arrhythmia was classified as supraventricular tachycardia, ectopic atrial tachycardia, atrial fibrillation / flutter, accelerated junctional rhythm, junctional ectopic tachycardia, ventricular tachycardia, or ventricular fibrillation . Evidence of chest pains or headache during milrinone use were collected from the patient's chart . Potassium levels and platelet counts 24 to 48 hours after starting milrinone infusion were estimated . Changes in hemodynamic parameters and echocardiographic data were analyzed using a pairwise comparison test and the wilcoxon signed - rank test . The frequency of hypokalemia and thrombocytopenia after milrinone infusion was analyzed using the mcnemar test . Univariate and multivariate logistic regression analysis was used to evaluate the relationships between diverse variables, including the practical use of milrinone and the development of arrhythmia . For statistical analyses, descriptive data are presented as means and standard deviation, whereas categorical variables are presented as proportions . Data manipulation and statistical analyses were performed with statistical package for the social sciences (spss) 19.0 for windows (spss inc ., somers, ny, usa) and excel 2010 (microsoft). The study protocol was approved by the institutional review board of seoul national university hospital and patient consent was waived due to the study's retrospective design . A retrospective analysis was conducted using data collected from patients who received milrinone treatment for 3 days at the seoul national university children's hospital from january 2005 to december 2012 . Patients were divided into two groups based on the indication for milrinone treatment: group a consisted of patients who received milrinone to prevent low cardiac output syndrome after cardiac surgeries whereas group b consisted of patients who received milrinone treatment due to acute heart failures from causes other than cardiac surgeries . Patients with the following conditions or those who underwent the following treatments were excluded from the study: 1) severe left ventricular outflow obstructive diseases such as aortic stenosis; 2) hypertrophic or restrictive cardiomyopathies; 3) milrinone administration immediately after cardiopulmonary resuscitation; 4) multi - organ failures; 5) simultaneous administration of 3 inotropes other than milrinone to increase blood pressure; 6) renal replacement therapies including peritoneal dialysis; 7) myocardial infarctions; 8) univentricular heart surgeries . However, patients receiving 3 inotropes were included if inotropes were added after an interval of> 2 hours consecutively . Patients' characteristics including age, sex, height, weight, and body surface area were recorded . The following parameters were analyzed to identify the clinical application of milrinone: 1) initial infusion rate; 2) maintenance continuous infusion rate; 3) total duration of milrinone therapy; 4) concomitantly infused inotropes . The operation and anesthesiology records of patients who had undergone cardiac surgeries were also studied to identify data related to anatomic diagnosis, type of surgery, time of cardiopulmonary bypass, and time of aortic cross - clamping . Systolic blood pressure, heart rate, and echocardiographic data (the left ventricular internal dimension at end - diastole, ejection fraction, and fractional shortening) obtained before or immediately after starting milrinone treatment were compared to the values prior to treatment cessation . In cases where appropriate echocardiographic data could not be obtained on the day milrinone treatment was discontinued, echocardiographic data obtained within a week of the treatment endpoint were used . The safety of milrinone was determined based on the occurrence of adverse events such as hypotension, arrhythmia, chest pain, headache, hypokalemia, and thrombocytopenia . Hypotension was defined as a sudden decrease in blood pressure within two hours after starting milrinone that necessitated the addition of inotropes or cessation of milrinone . Arrhythmia was classified as supraventricular tachycardia, ectopic atrial tachycardia, atrial fibrillation / flutter, accelerated junctional rhythm, junctional ectopic tachycardia, ventricular tachycardia, or ventricular fibrillation . Evidence of chest pains or headache during milrinone use were collected from the patient's chart . Potassium levels and platelet counts 24 to 48 hours after starting milrinone infusion were estimated . Changes in hemodynamic parameters and echocardiographic data were analyzed using a pairwise comparison test and the wilcoxon signed - rank test . The frequency of hypokalemia and thrombocytopenia after milrinone infusion was analyzed using the mcnemar test . Univariate and multivariate logistic regression analysis was used to evaluate the relationships between diverse variables, including the practical use of milrinone and the development of arrhythmia . For statistical analyses, descriptive data are presented as means and standard deviation, whereas categorical variables are presented as proportions . Data manipulation and statistical analyses were performed with statistical package for the social sciences (spss) 19.0 for windows (spss inc ., the study protocol was approved by the institutional review board of seoul national university hospital and patient consent was waived due to the study's retrospective design . In total, 2299 eligible admissions were identified during the study period . Of these admissions, a total of 730 admissions (684 patients) met the inclusion criteria and were selected for the present study . Twenty - seven patients received milrinone twice and one patient received milrinone thrice after serial cardiac surgeries . Most patients (715, 97.9%) received milrinone to prevent low cardiac output after cardiac surgeries . There were 715 admissions in group a and 15 admissions in group b. the male to female ratio was 5.6:1 . The mean age of patients at the time of milrinone infusion was 0.82 years (table 1). In total, 429 admissions (60%) in group a underwent cardiac surgeries at less than 3 months of age . The most common cardiac defect was ventricular septal defect in group a (42.4%), followed by tetralogy of fallot (11%) and coarctation of the aorta with ventricular septal defect (6.4%) (table 2). Accordingly, the most common type of surgery was closure of the ventricular septal defect, followed by repair of tetralogy of fallot and coarctoplasty . In group b, the duration of milrinone treatment ranged from 3 to 64.4 days . In 149 (20.4%) two or more inotropes other than milrinone were used in 425 (59.4%) admissions . The most commonly infused inotrope in group a was dopamine (83.4%), whereas all patients in group b received dobutamine (table 3). Systolic blood pressure, fractional shortening, and ejection fraction after milrinone treatment were significantly (p<0.05) increased in group a when compared to values obtained immediately after cardiac surgeries . However, there was no significant difference in systolic blood pressure, fractional shortening, and ejection fraction before and after milrinone treatment in group b. in both groups, heart rate was significantly (p<0.05) reduced after milrinone use (table 4). There were no reports of adverse events in the majority of admissions (78.7%) (table 5). Although diverse arrhythmias occurred in 75 (10.3%) admissions, only 3 (0.4%) required reduction or discontinuation of milrinone infusion to alleviate arrhythmia . Two of them developed junctional ectopic tachycardia and one of them developed ectopic atrial tachycardia . One - third of patients in group b as well as approximately 10% of patients in group a developed arrhythmia . In total, 41 (58.6%) of the 70 arrhythmias in group a were classified as junctional ectopic tachycardia, the development of arrhythmia was not influenced by the initial infusion rate, maintenance continuous infusion rate, or duration of milrinone treatment . Based on univariate logistic regression analysis of group a data, the following parameters were associated with the development of arrhythmia: male gender, age 3 months, and cardiopulmonary bypass time 180 minutes . Multivariate analysis of group a data in - dicated that male gender and cardiopulmonary bypass time 180 minutes influenced the development of arrhythmia . However, the incidence rate did not increase significantly (p=0.390), which was 108/730 (14.4%) at the initial point and 118/730 (16.2%) at 24 to 48 hours after starting milrinone treatment . Thrombocytopenia did increase slightly but significantly (p=0.039), which occurred in 4/729 (0.5%) at the initial point and 12/729 (1.6%) at 24 to 48 hours after starting milrinone treatment . Most patients in group a with thrombocytopenia required only a few platelet transfusions after cardiac surgeries . Platelet count was increased and maintained 50000/dl spontaneously in 2 admissions, after 1 platelet transfusion in 6 admissions, and after 2 platelet transfusion in 2 admissions . The cardiopulmonary bypass time (249.272.8 minutes vs. 153.170.4 minutes; p=0.004) were significantly longer in group a patients with thrombocytopenia than in patients without thrombocytopenia . The initial infusion rate, maintenance continuous infusion rate, and duration of milrinone treatment were not associated with thrombocytopenia . Two patients in group b had a low platelet count due to additional medical illnesses such as hemophagocytic lymphohistiocytosis and fungal infection . In total, 2299 eligible admissions were identified during the study period . Of these admissions, a total of 730 admissions (684 patients) met the inclusion criteria and were selected for the present study . Twenty - seven patients received milrinone twice and one patient received milrinone thrice after serial cardiac surgeries . Most patients (715, 97.9%) received milrinone to prevent low cardiac output after cardiac surgeries . There were 715 admissions in group a and 15 admissions in group b. the male to female ratio was 5.6:1 . The mean age of patients at the time of milrinone infusion was 0.82 years (table 1). In total, 429 admissions (60%) in group a underwent cardiac surgeries at less than 3 months of age . The most common cardiac defect was ventricular septal defect in group a (42.4%), followed by tetralogy of fallot (11%) and coarctation of the aorta with ventricular septal defect (6.4%) (table 2). Accordingly, the most common type of surgery was closure of the ventricular septal defect, followed by repair of tetralogy of fallot and coarctoplasty . In group b, the duration of milrinone treatment ranged from 3 to 64.4 days . In 149 (20.4%) two or more inotropes other than milrinone were used in 425 (59.4%) admissions . The most commonly infused inotrope in group a was dopamine (83.4%), whereas all patients in group b received dobutamine (table 3). Systolic blood pressure, fractional shortening, and ejection fraction after milrinone treatment were significantly (p<0.05) increased in group a when compared to values obtained immediately after cardiac surgeries . However, there was no significant difference in systolic blood pressure, fractional shortening, and ejection fraction before and after milrinone treatment in group b. in both groups, heart rate was significantly (p<0.05) reduced after milrinone use (table 4). There were no reports of adverse events in the majority of admissions (78.7%) (table 5). Although diverse arrhythmias occurred in 75 (10.3%) admissions, only 3 (0.4%) required reduction or discontinuation of milrinone infusion to alleviate arrhythmia . Two of them developed junctional ectopic tachycardia and one of them developed ectopic atrial tachycardia . One - third of patients in group b as well as approximately 10% of patients in group a developed arrhythmia . In total, 41 (58.6%) of the 70 arrhythmias in group a were classified as junctional ectopic tachycardia, whereas the most common arrhythmia identified in group b was ventricular tachycardia . The development of arrhythmia was not influenced by the initial infusion rate, maintenance continuous infusion rate, or duration of milrinone treatment . Based on univariate logistic regression analysis of group a data, the following parameters were associated with the development of arrhythmia: male gender, age 3 months, and cardiopulmonary bypass time 180 minutes . Multivariate analysis of group a data in - dicated that male gender and cardiopulmonary bypass time 180 minutes influenced the development of arrhythmia . However, the incidence rate did not increase significantly (p=0.390), which was 108/730 (14.4%) at the initial point and 118/730 (16.2%) at 24 to 48 hours after starting milrinone treatment . Thrombocytopenia did increase slightly but significantly (p=0.039), which occurred in 4/729 (0.5%) at the initial point and 12/729 (1.6%) at 24 to 48 hours after starting milrinone treatment . Most patients in group a with thrombocytopenia required only a few platelet transfusions after cardiac surgeries . Platelet count was increased and maintained 50000/dl spontaneously in 2 admissions, after 1 platelet transfusion in 6 admissions, and after 2 platelet transfusion in 2 admissions . The cardiopulmonary bypass time (249.272.8 minutes vs. 153.170.4 minutes; p=0.004) were significantly longer in group a patients with thrombocytopenia than in patients without thrombocytopenia . The initial infusion rate, maintenance continuous infusion rate, and duration of milrinone treatment were not associated with thrombocytopenia . Two patients in group b had a low platelet count due to additional medical illnesses such as hemophagocytic lymphohistiocytosis and fungal infection . In the present study, we reviewed the current patterns of milrinone use that continued for 3 days . Of a total of 2299 eligible admissions in which milrinone was used, 730 (31.8%) admissions involved the use of milrinone for 3 days . Although the korean national insurance covers only 35 hours of milrinone use, we found that milrinone was frequently prescribed for a longer duration in actual settings, suggesting a significant off - label use . The frequent use of milrinone in pediatric patients undergoing heart surgery was supported by the findings of the prophylactic intravenous use of milrinone after cardiac operation in pediatrics (primacorp) trial, which demonstrated that low cardiac output syndromes could be prevented by high - dose milrinone infusion after pediatric cardiac surgeries.10) although the initial and maintenance milrinone infusion rates determined in the present study were lower than those previously reported in the primacorp study, our values were similar to those reported in other european practices.4)10) in terms of efficacy issue, fractional shortening and ejection fraction were both improved after milrinone infusion in group a patients, which is in consistent with previous findings.9)11) chang et al.9) reported that milrinone treatment in neonates with low cardiac output after congenital heart surgery improved the cardiac index without changing myocardial oxygen consumption . Duggal et al.11) documented that milrinone therapy improved the left and right myocardial performance index, a reliable index that reflects myocardial contractility . Our findings revealed no significant changes in fractional shortening and ejection fraction after milrinone infusion in group b, which is in contrary to previously reported data in adults.12) the severity of the disease in group b is likely to have some influence on the results . Seven (46.7%) of the 15 admissions in group b involved patients who died due to underlying cardiac disease itself, reflecting the severity of disease in group b. in terms of safety, several adult studies have shown detrimental effects of milrinone on hemodynamic stability in chronic heart failures . The prospective randomized milrinone survival evaluation13) trial revealed that oral administration of milrinone to patients with heart failure increased mortality and induced serious adverse cardiovascular events including hypotension . The outcomes of a prospective trial of intravenous milrinone for exacerbations of chronic heart failure trial reported that sustained hypotension requiring additional treatment was more common in patients who received milrinone infusion for exacerbation of chronic heart failure.14) however, in the present study, only 1 (6.7%) of 15 admissions in group b needed a reduction in milrinone dose due to a decrease in blood pressure, with the incidence of hypotension being lower than that reported previously in adult data . Furthermore, only 3 (0.4%) of the 715 admissions in group a experienced hypotension that required a reduction in milrinone infusion rate . This result was in consistent with previous pediatric data that reported an infrequent occurrence of hypotension in the milrinone treatment group after cardiac surgery.10) milrinone use has been known to be associated with postoperative arrhythmia in adults that increased mortality and morbidity after cardiac surgery.15) arrhythmia is a concern following milrinone use in the pediatric population as well . Smith et al.16) recently reported that approximately half of the pediatric patients receiving milrinone after cardiac surgery experienced arrhythmia . In the present study, various arrhythmias occurred in 70 (9.8%) of 715 admissions in group a. however, only 3 (0.4%) admissions required modification of milrinone infusion to manage arrhythmias . Our finding is in consistent with that from other studies,17)18) indicating that the incidence of arrhythmia due to milrinone use in pediatric patients is relatively low after cardiac surgery . Postoperative arrhythmia is associated with age, cardiopulmonary time, aortic cross clamping time, and type of repair.19)20) in the present study, the initial infusion rate, maintenance infusion rate, and duration of milrinone treatment were not associated with the development of arrhythmia . However, in consistent with the previous findings, a longer cardiopulmonary time affected the development of arrhythmia in group a (table 6). Junctional ectopic tachycardia is the most common type of arrhythmia after cardiac surgery.20) in the present study, the incidence rate of junctional ectopic tachycardia in group a was 5.7% . This result is in accordance with previous studies, which reported that the incidence rate of junctional ectopic tachycardia after cardiac surgery range from 1.4% to 14.7%.21)22) the relationship between junctional ectopic tachycardia and milrinone use has not been elucidated thus far.23) a study in adults showed that atrial arrhythmia developed more frequently in patients with chronic heart failure who received milrinone treatment.14) however, the independent cause - effect relationship of milrinone with arrhythmia in pediatric patients with heart failure has not yet been established . Five (33.3%) of the 15 admissions in group b involved patients who mainly developed ventricular arrhythmia . In the present study, there was no significant difference in initial infusion rate, maintenance infusion rate, or duration of milrinone treatment between patients with or without arrhythmia in group b. in fact, heart failure due to myocarditis or dilated cardiomyopathy itself increased the vulnerability of the patients to tachyarrhythmia and conduction disturbance.24) ichikawa et al.25) reported that 3 of 7 patients with fulminant myocarditis developed ventricular tachycardia or ventricular fibrillation . Friedman et al.26) also documented the high incidence rate of arrhythmia in pediatric idiopathic dilated cardiomyopathy . The incidence rate of thrombocytopenia in the present study was lower than that in previous studies . Ten (1.4%) of the 715 admissions in group a involved patients who developed thrombocytopenia after milrinone treatment, which required a few platelet transfusions . Ramamoorthy et al.17) previously reported that milrinone treatment resulted in a high incidence (58%) of thrombocytopenia (platelet count <100000/dl) that did not often necessitate platelet transfusions . Bishara et al.18) further documented that 12.7% of admissions developed thrombocytopenia with milrinone treatment . However, it is difficult to identify milrinone - induced thrombocytopenia, because various factors could confound the observations, including the cardiac surgery itself . Cardiopulmonary bypass is known to be associated with thrombocytopenia that results from hemodilution and mechanical disruption.27) in the present study, patients who developed thrombocytopenia had a longer cardiopulmonary bypass time and aorta - clamping time . Thus, patients with a more complex heart disease and longer bypass times during cardiac surgery would be more likely to develop thrombocytopenia . In contrast to amrinone, the first phosphodiesterase inhibitor used that was known to have adverse effect on platelets, milrinone did not alter platelet count or function in patients after cardiac surgery.28) the fact that two (13.3%) patients in group b developed thrombocytopenia could be due to complications such as hemophagocytic lymphohistiocytosis and fungal sepsis, rather than due to milrinone use . The utility of chronic continuous milrinone infusion for heart failure as bridge therapy to heart transplantation was proposed in a recent adult study.6) mcmahon et al.29) demonstrated that long - term support with milrinone was safe and effective in infants with cardiomyopathy awaiting heart transplantation . Outpatient continuous parenteral inotropic therapy, including milrinone, has also been used in inotropic - dependent children with advanced heart failure.30) although these studies used small patient populations, the incidence of complications was sufficiently low to indicate the safe and effective potential of milrinone use in infants and children with heart diseases . This study had a retrospective design which did not have a control group containing patients who did not receive milrinone . Moreover, all arrhythmias could not be included due to the study's retrospective nature . Furthermore, because the exclusion criteria of this study were relatively strict to appropriately clarify the efficacy and safety of milrinone use and reduce confounding factors, the patients who would be more susceptible to milrinone use could not be included . In surgically treated patients with congenital heart disease, we should have also considered the natural improvement of cardiac function after the corrective operation, which could be a confounding factor for evaluating the efficacy of milrinone use in this study . Finally, only a few echocardiographic data including ejection fraction and fractional shortening were used to evaluate milrinone efficacy due to the retrospective study design . If other echocardiographic data, such as tissue doppler imaging findings, were available, a more accurate evaluation of the hemodynamic effects of milrinone would have been possible . This is the largest retrospective study, to date, to evaluate the safety and efficacy of long - term milrinone use (3 days) in pediatric patients with cardiac diseases . Such long - term milrinone use is common in actual clinical practice, and milrinone is generally used for> 35 hours in infants and children with heart disease . Our data revealed that the use of milrinone for 3 days was effective in preventing low cardiac output after cardiac surgery when combined with other inotropes . This study had a retrospective design which did not have a control group containing patients who did not receive milrinone . Moreover, all arrhythmias could not be included due to the study's retrospective nature . Furthermore, because the exclusion criteria of this study were relatively strict to appropriately clarify the efficacy and safety of milrinone use and reduce confounding factors, the patients who would be more susceptible to milrinone use could not be included . In surgically treated patients with congenital heart disease, we should have also considered the natural improvement of cardiac function after the corrective operation, which could be a confounding factor for evaluating the efficacy of milrinone use in this study . Finally, only a few echocardiographic data including ejection fraction and fractional shortening were used to evaluate milrinone efficacy due to the retrospective study design . If other echocardiographic data, such as tissue doppler imaging findings, were available, a more accurate evaluation of the hemodynamic effects of milrinone would have been possible . This is the largest retrospective study, to date, to evaluate the safety and efficacy of long - term milrinone use (3 days) in pediatric patients with cardiac diseases . Such long - term milrinone use is common in actual clinical practice, and milrinone is generally used for> 35 hours in infants and children with heart disease . Our data revealed that the use of milrinone for 3 days was effective in preventing low cardiac output after cardiac surgery when combined with other inotropes.