Background. Previous studies that assessed the role of birth weight and gestational age in the risk of asthma have been conflicting. Objectives. To examine the association between birth weight and gestational age and symptoms of asthma. Patients and Methods. Subjects were 6656 school children of ages 6-7 and 13-14 years from urban districts of Mazandaran, Iran. ISAAC questionnaires were used. Results. There was an increased risk of “wheeze ever” in both age groups with birth weight under 2.5 kg and in all subgroups of low birth weight (LBW). Birth weight more than 3.5 kg was associated with lower risk of “severe asthma” in age group 6-7 years. With respect to gestational age, higher risks of “wheeze ever,” “asthma ever,” and “night cough in the past 12 months” were found in age group 13-14 years born before 37 weeks and the risk of “severe asthma” was higher in younger group (6-7 years). A lower risk of "asthma ever" was also found in 6-7-year-old children and 13-14-year-old girls who were born after 40 weeks. Conclusions. This study showed that there is a direct relation between “wheeze ever” and LBW and an inverse relation between risk of “severe asthma” and birth weight more than 3.5 kg. Asthma is a known global public health issue. Its prevalence in different countries ranges from 0.7 to 18.4% . The cauMaternal infections –11, pregIn some studies, the relationship between birth weight and prematurity has been proposed as a perinatal risk factor for asthma –27. For One obstacle to the investigation of population differences had been the lack of a suitable and generally accepted method of measuring the prevalence and severity of asthma in children which could be used worldwide. Another obstacle was the absence of a coordinated research program to obtain and analyze comparative data. The International Study of Asthma and Allergies in Childhood (ISAAC) program was developed in 1991 to evaluate the prevalence and severity of asthma, rhinitis, and eczema in children living in different centers, to describe the data and make it be comparable, and to provide a framework for further etiological researches about factors affecting these diseases.There is large variations in the prevalence of symptoms of asthma and allergic disease throughout the world (more than 20-fold between centers) and between people of similar genetic origin living in different environments; it was concluded that the environmental factors are the cause of these large variations such that the prevalence of “wheeze in the past 12 months” is 2.1 to 32.2% in age group 13-14 years and 4.1 to 32.1% in age group 6-7 years. The highest percentage belongs to English speaking countries and Latinos .A systematic review in 2014 which is based on the results of ISAAC phase 3, concerning the correlation between birth weight and the prevalence of allergic disease's symptoms in children of 6-7 years of age, showed that low birth weight (<2.5 kg) was associated with an increased risk of symptoms of asthma . High birth weight (birth weight ≥ 4.5 kg) was not associated with any of these outcomes [This study has evaluated the association between gestational age  and birth weight with the prevalence and severity of asthma symptoms with the use of ISAAC questionnaires, which has been done in 6-7- and 13-14-year-old children in primary schools, Mazandaran, Iran.Participants were students from urban schools of Ramsar and Tonekabon . They were of two different age groups (6-7 and 13-14 years). All subjects had written consent from their parents. ISAAC written questionnaires for prevalence were filled by parents of younger age group, equivalent questionnaires were filled by children of age group 13-14, and the risk factor questionnaires were filled by parents in both age groups.Symptoms of asthma and wheezing were evaluated as “wheeze ever,” “wheeze in the past 12 months” (current wheeze), “asthma ever,” “wheeze during or after exercise in the past 12 months,” and “night cough in the past 12 months.”Four or more attacks in the past 12 months.Sleep disturbance in one or more nights per week due to difficulty in respiration in the past 12 months.Difficulty in pronouncing two-word phrases in the past 12 months.A sample size of 3000 was chosen, which gives the following power: (1) prevalence of wheezing: if the true one-year prevalence of wheezing is 30% in one center and 25% in another center, with a sample size of 3000, the study power to detect this difference will be 99% at 1% level of significance; (2) severity of wheezing: if the true one-year prevalence of severe asthma is 5% in one center and 3% in another center with a sample size of 3000, the study power to detect this difference will be 90% at 1% level of significance.Diagnosis of severe asthma was based on the presence of any of the following criteria:Descriptive results were reported using frequency and percent. Odds ratios (ORs) were calculated with generalized linear models, with a binomial distribution and logit link, adjusted for sex to fit for all participants. ORs more than 1 show more risk in contrast to reference group. The symptoms were modeled as function of birth weight/gestational age, and sex and their interaction. If the sex-by-treatment interaction was not statistically significant, the model was refitted with only main effects for treatment. The reference category was 2.5 to 3.5 kg for birth weight and 37 to 40 weeks for gestational age. First models were fitted as a function of birth weight by three groups  and to know what happen below 2.5 kg. The models were refitted as function of birth weight by five groups . The significance level was 0.05. The sample size was at least 3000 for each age group which was chosen based on ISAAC manuals.6656 students participated in this study: 3102 belong to age group 6-7-years and 3554 belong to age group 13-14 years. Age group 6-7 years consists of 46.7% boys and 53.3% girls and age group 13-14 years consists of 51.9% boys and 48.1% girls. In total and without gender and age discrimination, 19.8% of the students reported “wheeze ever” and 28.1% reported “wheeze in the past 12 months (current wheeze).” Birth weight distribution exhibited that 8.7% of 6-7-year-old and 16.6% of 13-14-year-old age groups were under 2500 g. 30.6% of 6-7-year-old and 33% of 13-14-year-old age groups had birth weight over 3.5 kg see .Based on descriptive statistics analysis, the prevalence of “wheeze ever” and “wheeze in the past 12 months” in age group 6-7 years and in both genders in this group was higher than age group 13-14 years. Also, in children with birth weight under 1.5 kg (and in both genders in this group), the prevalence of “wheeze after exercise in the past 12 months” and “night cough in the past 12 months” was higher than other birth weight groups. The prevalence of studied symptoms with respect to birth weight and gender is shown in p = 0.01), but the results showed a higher OR for females in the same group (OR = 1.315).Odds ratios for “wheeze ever,” “wheeze in the past 12 months,” and “night cough in the past 12 months” in children with birth weight lower than 2.5 kg in this age group were 1.32, 1.01, and 1.32, respectively. Odds ratios for the same symptoms in children with birth weight more than 3.5 kg are 1.12, 1.11, and 1.09, respectively. Higher odds are found for males rather than females. However, odds ratio of “asthma ever” and “wheeze during or after exercise in the past 12 months” in children with birth weight lower than 2.5 kg is 0.44 and 0.67, respectively, but in group with birth weight higher than 3.5 kg, these ORs were slightly higher than that of the reference group. Similar results were obtained with respect to gender in group with birth weight higher than 3.5 kg. “Severe asthma” was the only variable with OR lower than the reference group and was significantly lower than the reference in the male group  and “wheeze in the past 12 months”  were significantly higher than the reference and higher than two other subgroups. This difference is also more strongly observed in the female group with birth weight lower than 1.5 kg in comparison with two other subgroups. Higher odds ratio of “night cough in the past 12 months” was observed in subgroup under 1.5 kg as compared to other groups. Similar results were detected with respect to gender  and “night cough in last 12 months”  were considerable. Females had higher odd ratios than males except in “asthma ever” symptom.In group with birth weight higher than 3.5 kg, ORs of all symptoms were higher than the reference; however, with gender consideration, ORs of some symptoms were lower than the reference group. In female, ORs of “wheeze ever,” “asthma ever,” “wheeze during or after exercise in the past 12 months,” and “night cough” were 1.37, 2.44, 2.10, and 1.73, respectively, which were higher than ORs in male group.With respect to subcategorization of group with birth weight under 2.5 kg, it was observed that the ORs of all symptoms except “asthma ever” were higher than the reference in all subgroups, especially with birth weight 2 to 2.5 kg. ORs of all symptoms were lower than the reference in males with birth weight of 1.5 to 2 kg. In males with birth weight under 1.5 kg, odds ratios of “wheeze during or after exercise in the past 12 months,” “asthma ever,” and “night cough in last 12 months” were higher than the reference .In age group 6-7 years, “wheeze ever” ORs were lower than the reference in groups with gestational age lower than 37 weeks and more than 40 weeks; conversely, “severe asthma” ORs in both groups were 3.65 and 2.06 (higher than reference group). ORs for “wheeze in the past 12 months,” “wheeze during or after exercise in the past 12 months,” and “night cough in the past 12 months” were higher in group with gestational age more than 40 weeks with 1.11, 3.04, and 1.45, respectively. Odds ratio for “asthma ever” was lower than the reference; meanwhile, “asthma ever” OR for group with gestational age under 37 weeks was higher than reference group . Also, ORs for “asthma ever” and “night cough in the past 12 months” were higher than the reference. Generally, ORs for all symptoms were higher in males than females. For women, ORs of “asthma ever,” “wheeze during or after exercise in the past 12 months,” and “night cough in the past 12 months” were lower than the reference  [p = 0.030) and “night cough in the past 12 months” (p = 0.004) were significantly higher than reference and, like low birth weight (under 2.5 kg), it is accompanied with a higher rate of asthma and its severity in age group 13-14 years. Previous studies did not examine the role of low birth weight in age group 13-14 years.According to a meta-analysis of Auckland University from the results of ISAAC studies in 69 centers from 38 countries on more than 1000 population with more than 60% response rate, despite all the differences between centers, children with birth weight lower than 2.5 kg carried a higher risk of developing asthma symptoms in age group 6-7-years. In this study, adjusted OR for “severe asthma” was greater than “wheeze in the past 12 months”, and no significant variation was seen between sexes (interaction  > 0.05) . In our In a cross-sectional study, based on the diagnostic criteria of ISAAC for evaluation of allergic disease symptoms in 3-year-old children with history of preterm birth or low birth weight in Japan, no significant association was found between low birth weight (under 2.5 kg) and the prevalence of wheezing and asthma. In the aforementioned study, weight group under 2.5 kg was not divided into smaller subgroups . In our In a cohort study in Denmark, Sweden, and Finland, the risk of hospitalization with severe asthma attack was considerably higher in children with low birth weight but this relationship was not seen in children with a history of preterm birth. This study did not evaluate the severity of asthma attacks, but the results of our study showed an association between severe asthma (based on asthma control) and birth weight less than 2.5 kg, in 13-14-year age group and girls of 6-7-year age group .p = 0.42). After propensity score matching and considerable decrease of covariate imbalance and observing the same risk in both groups, it was concluded that there is no association between the incidence of asthma and birth weight [In a retrospective cohort study, the incidence of asthma was compared in children with birth weight above 2500 g and with birth weight below 2500 g. 6.7% of children with birth weight under 2500 g and 5.4% of children with birth weight more than 2500 g showed symptoms of asthma (h weight . In the Significantly lower ORs for “severe asthma” have been shown in the 6-7-year group with birth weight above 3.5 kg (sig = 0.044). In a cohort study which evaluated the relationship between high birth weight and risk of asthma before 6 years of age in Toronto, the risk of asthma has been shown to be reduced with high birth weight ; nevertheless, the risk of asthma is slightly increased with substantial high birth weight (more than 6 kg) .On the other hand, a cohort study in Finland evaluated the relationship between high birth weight with chances of atopic symptoms including asthma, until 16 years of age (based on the physician's diagnosis); results demonstrated a higher risk of asthma in children with birth weight above 4510 g and suggested that increased risk of asthma is caused by escalation in risk of atopic disorders in obese children .A meta-analysis, which studied articles from 1990 to 2013, reported no increase in risk of asthma in children with birth weight more than 4000 g . FurtherISAAC study from Auckland University showed that high birth weight does not increase the risk of asthma symptoms and suggested that other studies which showed increase in ORs of asthma are caused by bias. This study did not find an increase in asthma symptoms in 6-7-year-old children with high birth weight; however, an increased risk of “night cough in the past 12 months” and “wheeze in the past 12 months” (current wheeze) was found in 13-14-year-old children with high birth weight. This discrepancy could be the result of the difference in weight grouping in our study (>3.5 kg) versus ISAAC (>4 kg). It could also be a result of the different studied age groups by ISAAC (6-7 years old) and our study (6-7 and 13-14 years old). In other words, high birth weight could cause higher risk of asthma symptoms in adolescence, but not in childhood. A decreased risk of “asthma ever” and exercise asthma in girls of 13-14 years with gestational age of more than 40 weeks has been found.p < 0.05) and the associations of lower birth weight with childhood asthma were largely explained by gestational age at birth [A meta-analysis for 147,252 children of 31 birth cohort studies to determine the associations of birth and infant growth characteristics with the risks of preschool wheezing (1–4 years) and school-age asthma (5–10 years) showed that lower gestational age at birth was associated with higher risks of preschool wheezing and school-age asthma (at birth . An incrThis study has also provided evidence about increased risk of “severe asthma” and “wheezing during or after exercise in the past 12 months” in the age group of 6-7 years with gestational age of more than 40 weeks and also an increased risk of “asthma ever” and “wheeze ever” in the age group of 13-14 years.This study showed that there is no linear association between birth weight or gestational age with severity of asthma, but the risk of “wheeze ever” is increased in both age groups with LBW and with prematurity in 13-14-year age group. There was also an increased risk of “severe asthma” in 6-7-year-old children with a history of prematurity.


The nutritional care provided to moderately premature babies is poorly studied. For a large cohort of such babies, we aimed to describe: nutrition practice intentions, comparison of the intended with the actual practice, compliance of actual practice to current nutrition guidelines, and postnatal growth.w 36PCA/DC) were analysed by multivariate linear regression.A questionnaire was sent out to 29 neonatal intensive care units in France, in order to address practice intentions. In the same units, retrospective patient’s data were collected to assess actual practice, compliance to nutrition guidelines and infant postnatal growth. The cumulative nutritional deficit during the two first weeks of life was calculated and variables associated with ΔZ-score for weight at 36 weeks postconceptional age/discharge , 93 % of infants had parenteral amino acids (AA), at an intake ≥ 1.5 g/kg in 27 % of cases. Lipids were started at ≤ DOL2 in 47 % of infants. There was a divergence between the intended and the actual practice for both AA and lipids intake. The AA and energy cumulative deficit (DOL1 to DOL14) were respectively 10.9 ± 8.3 g/kg and 483 ± 181 kcal/kg. Weight Z-score (mean ± SD) significantly decreased from birth (−0.17 ± 0.88) to 36 weeks PCA/DC (−1.00 ± 0.82) (p < 0.0001), and the extra-uterine growth retardation (EUGR) rate at 36 weeks PCA/DC was 24.2 %. Independent variables associated with ΔZ-scoreNutrition intake was not in compliance with recommendations, and the rate of EUGR was considerable in this cohort. Efforts are needed to improve adherence to nutrition guidelines and growth outcome of moderately preterm infants. In the last decades, several studies demonstrated serious cumulative nutritional deficit and extra-uterine growth retardation (EUGR) in preterm infants during the first weeks of life –3.More recently, the causes of this growth failure have been explored and in part identified. The implementation of continuous education, the carrying out of surveys to focus on trends in clinical practice , 5 and tAll this literature relates to extremely or very low birth weight (VLBW) infants and very little is known about the nutritional care of the more “healthy” but still immature babies, born between 30 and 33 weeks of gestational age (GA), even though they account for 15 % of preterm life births and 30 % of neonatal intensive care unit (NICU) admissions.Some reports have demonstrated that these infants, too, fail to achieve intrauterine growth rates during hospital stay and that significant variations in feeding practices and growth outcomes exist among NICUs , 9. HoweInfants within this cohort are generally less “sick” when compared with their very preterm counterparts and they have more subtle feeding issues and complications. Moreover, they often require a limited technological support and the choice of avoiding the central venous line (CVL) insertion – in order to reduce the possible associated complications - may limit the supply of the recommended protein and caloric intake, especially during the transitional period .So, the determinants of postnatal growth and the factors influencing the adequacy of nutrition support in moderately preterm babies may be different from those of VLBW infants and deserve a better understanding.The aims of this study were the following: 1) to describe nutrition practice intentions in a large cohort of moderately premature babies; 2) to compare nutrition practice intentions to actual practice; 3) to describe the adherence of units’ actual practice to current nutrition guidelines; 4) to measure the growth outcomes during hospital stay in this cohort.A survey was carried out in 2014 in French mainland and overseas departments and territories. It was designed to include a heterogeneous group of preterm infants born between 30 and 33 weeks of gestation and cared for in tertiary NICUs or secondary (IIB) level nurseries. For the purpose of this study, infants born between 30 to 33 weeks of gestation were defined “moderately premature babies". The survey consisted of one-page questionnaire and an electronic file to collect patients’ data. The aims of the questionnaire and of the electronic file were respectively to address the unit nutrition practice intentions and to describe the actual adherence to current nutrition guidelines.The questionnaire was addressed to the senior physician of each unit or to a delegated colleague having clinical experience of neonatal intensive care, and it consisted of multiple choice and open-ended questions. Specifically for infants born at 30–31 and at 32–33 weeks of gestation, the questionnaire assessed the following variables: whether written nutrition guidelines were available in the unit, whether parenteral nutrition (PN) was considered since the day of birth, the type of venous access chosen in case of PN, postnatal day on which parenteral AA and lipid intake was started, AA starting dose, postnatal day on which enteral feeding was started, enteral feeding advancement and fortification strategy.PN was defined as “intravenous nutrition given through a central or peripheral line and containing at least both dextrose and nitrogen”, full enteral feeding as “enteral feed given as sole nutritional source”. Day of life (DOL) 1 was defined as the day of birth.In each centre, respondents were asked to record data from the last consecutive 12 patients on the electronic file  being at 36 weeks postconceptional age (PCA) or discharged from the unit.Exclusion criteria were: major or digestive congenital anomalies, outborn, transfer to other hospital within DOL14 and death within the first week of life.The identity of the respondent to the questionnaire and that of the centre’s electronic file were both blinded for the analysis to all authors. Patients’ records were anonymous.Data recording for this study was approved by the National Committee for data protection .This study was approved by the institutional medical research ethics committee . According to French legislation, written parental consent was not needed for this study.Demographic features and in-hospital morbidities were collected. The following data were recorded on DOL1, 3, 7, 14 and at 36 weeks PCA or at discharge (DC) (when before 36 weeks PCA): weight, parenteral  and enteral intake (ml/kg/d of human or given formula milk), whether a central or peripheral venous line was inserted.Mean human milk content was assumed to be 64 kcal⁄dl, 1.4 g/dl of protein, 3.2 g/dl of fat and 7.0 g⁄dl of carbohydrate. Human milk fortifier and preterm infant formula compositions were based on the product’s labelled nutritional content.Head circumference and length were measured at birth and at 36 weeks PCA/DC. Finally, information was retrieved on total days of PN on central and peripheral venous line, DOL on which trophic or enteral feeding was started, DOL on which milk fortification was started, type and amount of milk fortifier, DOL of full enteral feeding and maximal postnatal weight loss.Nutrition practice intentions were compared to the actual practice with regard to the following variables: type of venous access at birth, DOL of initiation of enteral feeding, DOL of initiation for parenteral AA and lipids and AA starting dose.Compliance to nutrition guidelines was measured according to current European recommendations  with theThe cumulative AA (g/kg) and energy intake  (DOL1 to DOL14) was estimated according to the formula:r2 = 0.9729; p < 0.001 and 0.9648; p < 0.001 respectively for AA and energy intake).The formula was elaborated based on the assumption that a linear progression of intake occurred between DOL1 to DOL3, DOL3 to DOL7, and DOL7 to DOL14, and it was validated in a subgroup of the cohort study: in 48 infants (from 4 units) the AA and energy intake was collected daily from DOL1 to DOL14 and the actual cumulative intake (DOL1 to DOL14) was correlated to the cumulative intake estimated by the formula by a linear regression procedure. The model showed a very good regression coefficient  for AA and energy was calculated as following:For the purpose of some analyses, infants and centres were split into tertiles of respectively AA cumulative intake and mean AA cumulative intake.Z-score [mean and standard deviation (SD) for GA] for weight, length and head circumference was calculated at birth and at 36 weeks PCA/DC, according to the reference .th percentile according to the reference [Infants were considered SGA when the sex- and age-adjusted weight at birth was below the 10eference .th percentile according to the reference [They were defined EUGR when they were not born SGA and the sex- and age-adjusted weight at birth and at 36 weeks PCA/DC was below the 10eference .w 36PCA/DC).Simple and multiple linear regressions were performed to investigate variables associated with the delta Z-score for weight at 36 weeks PCA/DC  of the model was calculated, in order to choose the best-fit equation for the data set.Potential cofounders were selected among antenatal , clinical  and nutritional  factors. These were included in the multivariate model only if they were significant at a χ2-test (or Fisher’s exact test) for categorical variables; the ANOVA test was used for parametric variables and the Mann–Whitney U test for non-parametric continuous variables.Comparisons between groups were performed using p values <0.05 were considered statistically significant.All statistical analyses were carried out using the MedCalc. ver. 12.3.0.0 statistical software package  and A total of 29 units were surveyed. Four units were secondary excluded as they only provided responses to the questionnaire without feedback of patients’ data.So, finally, 25 units were included in the analysis (23 III level NICUs and 2 IIB level nurseries).Data of 276 patients were analysed. Population characteristics are presented in Table Table Among the 276 infants, 92 % had a PN and 76 % a CVL for PN. The mean duration of PN on CVL was 9.0 ± 8.1 days.The average time of initiation of enteral feeding was 1.7 ± 1.2 DOL. Feeding volume (mean ± SD) was advanced from 9 ± 12 ml/kg/d on DOL1 to 38 ± 26 ml/kg/d on DOL3, reaching 87 ± 48 ml/kg/d at DOL7 and 140 ± 50 ml/kg/d by DOL14. The average day of full enteral feeding was 11.7 ± 6.9 DOL. A total of 73 % of all study subjects received human milk during their hospital stay and 42 % were at least partially breastfed at 36 weeks PCA/DC. The average time of initiation of milk fortifier was 9.9 ± 5.5 DOL. The global rate of milk fortification was 65 % and 36 % of all infants received fortified feeding at 36 weeks PCA/DC.Parenteral AA were started on DOL1 in 93 % of infants, the administered dose at DOL1 was ≥ 1.5 g/kg in 27 % and lipid administration was started at ≤ DOL2 in 47 %. The results on the AA and lipid administration practice and the comparison of the intended and actual practice according to groups of GA are presented in Table For the all population study, the AA intake (g/kg/d) was 1.1 ± 0.7 at DOL1; 2.9 ± 0.9 at DOL7, and the energy intake  94 ± 18 at DOL7. The cumulative AA (g/kg) and energy intake  were respectively 37.0 ± 8.3 and 1201 ± 166 and the correspondent cumulative deficits were 10.9 ± 8.3 and 483 ± 181.Table th percentile according to the reference13 was 36.7 % (12.5 % of infants being born SGA and 24.2 % having EUGR).For the all population study, the maximal postnatal weight loss was 8.3 ± 3.9 %. At 36 weeks PCA/DC the rate of babies whose weight was below the 10w 36PCA/DC at the univariate analysis were: infant tertile for AA cumulative intake, centre tertile for mean AA cumulative intake, cumulative caloric intake, DOL of full enteral feeding, SGA, GA, labour, Apgar ≤ 3 at 1 min of life, phototherapy (data not shown). Variables associated with ΔZ-score at 36 weeks PCA/DC at the multivariate analysis are detailed in Table Variables associated with ΔZ-scoreVariables associated with cumulative deficit of protein intake (DOL 1–14) at the multivariate analysis are detailed in Table w from birth to 36 weeks PCA/DC in this vulnerable population.This study shows the poor postnatal growth of a moderately premature population, demonstrating that both EUGR rate and cumulative nutritional deficit are considerable during the first weeks of life in infants born 30 to 33 weeks of gestation. The main result is that a better nutritional intake may significantly reduce the ΔZ-scoreCritical illness, insufficient early PN support, lack of education and training in PN prescription have been proven responsible for the growth retardation acquired during hospitalization of extremely and VLBW infants , 14–16.To our knowledge, this is the first time that the impact of all these factors on the growth outcome has been explored for the specific population of more healthy but still immature preterm babies. Indeed, several clinical and nutritional variables potentially influencing the degree of growth failure have been investigated by this report.Studies in extremely low birth weight infants have underlined that insufficient early PN support and EUGR are a serious problem, especially for neonates who are small, immature and critically ill, and that they are independently associated to acute morbidities and illness conditions  .Recently, Santerre and Rigo , 7 have Their observation was assessed in a cohort of extremely and VLBW infants, but based on our results, it may also apply to a more healthy population of larger babies and more advanced GA.Our study has also documented that shorter time to achieve full enteral feeding was significantly associated with a more favourable growth outcome. This is consistent with the results of Ehrenkranz et al. , in a prInteresting also, in our study, cumulative protein deficit appears significantly lower in babies with lower gestational ages and with intrauterine growth retardation, it is inversely associated with centre tertile for mean AA intake and with the use of CVL on DOL1, and it is not influenced by prematurity-related morbidities.According to all the above data, differences in growth in our population would more clearly reflect differences in nutritional care and may reveal more effective feeding strategies among centres. This result has interesting implications for clinicians and NICU policies.In an original way, this study aimed to evaluate the adherence of the intended and the actual practices to the recommended PN guidelines for preterm infants, even if the information was collected within the limits of the starting administration of AA and lipids. It is worthwhile to note that, whether both the practices for DOL of starting AA were in compliance with the established PN guidelines, this was not the case for the AA starting dose, or for lipids administration. This is an important point to be addressed in the course of continuous education strategies, because, as noticed in a recent report from Fischer and coll. , early pFinally, our results somehow show a discrepancy between what physicians intend to prescribe and what they do really provide to preterm babies, and this leads to the following considerations: the first one regards the methodological limitation of using surveys for the assessment of nutritional protocols, as already highlighted by previous reports , 15, 19;w at 36 weeks PCA/DC has a quite poor general coefficient, suggesting that other variables not considered in the analysis may account for explaining the growth outcome in the studied population. Finally, the present investigation was not designed to explore enteral feeding practices in detail in terms of fortification strategies, feeding techniques and breastfeeding polices, which might produce differences in growth outcome, as illustrated by other studies [The present study has several limitations. The major one is undoubtedly that our data may not be considered as representative of the entire population of the moderately premature babies targeted by the survey, especially with regards to results of intention-to-treat practices, as questionnaires were not distributed on a national basis, contrary to most surveys , 15. A f studies , 21.The results of our report have interesting implications for clinicians and NICU policymakers. As already demonstrated for extremely and VLBW infants, our study allows to conclude that the nutrition delivered to infants born 30–33 weeks gestation is often not in compliance with international guidelines, and that this is associated with considerable rates of EUGR.The reasons for suboptimal AA and total calories delivery in this specific population mainly reside on unit nutrition practice and feeding strategies, this making that the growth outcome may be improved.Factors influencing EUGR in moderately premature babies are not fully elucidated, and further studies are needed to identify practices associated with better growth in this quite healthy, but still vulnerable infant population.


To verify whether body mass index (BMI) classification proposed by the Institute of Medicine (IOM) is valid in Japanese women.A study was conducted in 97,157 women with singleton pregnancies registered in the Japan Society of Obstetrics and Gynecology (JSOG) Successive Pregnancy Birth Registry System between January 2013 and December 2013, to examine pregnancy outcomes in four groups stratified by pre-pregnancy BMI category according to the 2009 criteria recommended by the Institute of Medicine (IOM). The groups comprised 17,724 underweight women with BMI <18.5, 69,126 normal weight women with BMI 18.5–24.9, 7,502 overweight women with BMI 25–29.9, and 2,805 obese women with BMI ≥30. The pregnancy outcomes were also compared among subgroups stratified by a gestational weight gain below, within, and above the optimal weight gain.The higher the pre-pregnancy BMI, the higher the incidences of pregnancy-induced hypertension, gestational diabetes mellitus, macrosomia, cesarean delivery, postpartum hemorrhage, and post-term birth, but the lower the incidence of small for gestational age (SGA). In all pre-pregnancy BMI category groups, excess gestational weight gain was associated with a higher frequency of large for gestational age and macrosomia; poor weight gain correlated with a higher frequency of SGA, preterm birth, preterm premature rupture of membranes, and spontaneous preterm birth; and optimal weight gain within the recommended range was associated with a better outcome.The BMI classification by the IOM was demonstrated to be valid in Japanese women. The patient records was anonymized and de-identified prior to analysis. The present study was a retrospective investigation of women with singleton pregnancies who were included in the Japan Society of Obstetrics and Gynecology (JSOG) registry system. Approximately 280 secondary and tertiary hospitals participated in the JSOG Successive Pregnancy Birth Registry System, which collected information on successive deliveries occurring at gestational week 22 or later. A total of 186,235 women were registered in the system between January 1, 2013 and December 31, 2013. Women with concomitant hypertension or diabetes as the underlying disease, with a history of cervical conization, who delivered a newborn with congenital anomalies, or whose data were unknown, were excluded. After the exclusion, 97,157 women were included in the study .2), the pre-pregnancy overweight group (BMI = 25–29.9 kg/m2), the pre-pregnancy normal weight group (BMI = 18.5–25 kg/m2), or the pre-pregnancy underweight group (BMI <18.5 kg/m2).Based on the 2009 IOM guidelines, 97,157 women were classified into the pre-pregnancy obese group , gestational diabetes mellitus (GDM), macrosomia, small for gestational age (SGA), large for gestational age (LGA), preterm birth, spontaneous preterm birth, preterm premature rupture of membranes (PPROM), induced preterm birth, cesarean delivery, severe postpartum hemorrhage, and post-term birth; the incidence of these outcomes was compared among the groups and analyzed. PIH was defined as a case in which hypertension (systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg) developed after 20 weeks of gestation. GDM  was diagnosed when at least one of the following was found: fasting blood glucose level of ≥92 mg/dL, blood glucose level at 1 h of ≥180 mg/dL, blood glucose level at 2 h ≥153 mg/dL. Macrosomia was defined as a neonatal birthweight 4,000 g or more. SGA was defined as a neonatal birthweight below the 10th percentile of the reference curves of birthweight for gestational week. LGA was defined as a neonatal birth weight above the 90th percentile of the reference curves of birth weight for gestational week. Induced preterm birth was defined as preterm delivery by cesarean section or induction of labor due to maternal complications, such as preeclampsia or non-reassuring fetal status. Spontaneous preterm birth was defined as other than preterm birth medically indicated by cesarean section or labor induction. Postpartum hemorrhage with vaginal delivery (PPH with VD) was defined as an estimated blood loss of 500 mL or more; postpartum hemorrhage with cesarean delivery (PPH with C/S) was defined as an estimated blood loss of 1,000 mL or more. Post-term birth was defined as a delivery at ≥42 and 0/7ths weeks of gestation. Gestational age was determined based on the last menstrual period. If gestational age according to the last menstrual period differed by more than 7 days from that based on ultrasonography at less than 11 weeks, the latter was used to assign a gestational age [Next, in order to investigate the relationship between weight gain during pregnancy and the pregnancy-delivery outcome, a stratified analysis was performed in three subgroups of gestational weight gain below, within, and above the optimal range, based on the IOM-recommended 2009 guidelines, to compare the frequency of PIH, GDM, SGA, high-fat diet, preterm birth, spontaneous preterm birth, PPROM, induced preterm birth, PPH with VD, PPH with C/S, macrosomia, and post-term birth in each group by pre-pregnancy BMI categories of underweight, normal weight, overweight, and obese.Data were expressed as means ± standard deviation or frequencies (percentages). SPSS Statistics software version 23  was used for the statistical analyses. One way analysis of variance (ANOVA) for continuous and a chi-square test for categorical data were used to compare the maternal characteristics and pregnancy outcomes among the four groups, with Bonferroni correction for multiple comparisons. A p value of <0.05 was considered statistically significant. Logistic regression analysis was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs), adjusting for confounding variables, including maternal age, maternal height and parity. Generalized estimating equations (GEE) for logistic regression was used to adjust for clustering of deliveries by hospitals.Of 97,157 pregnancies, the pre-pregnancy underweight group accounted for 18.2% , the pre-pregnancy normal weight group for 71.1% , the pre-pregnancy overweight group for 7.7% , and the pre-pregnancy obese group for 2.9% . Tables In the underweight and normal weight groups, the incidences of PIH, LGA, and macrosomia were significantly higher with increasing gestational weight gain (p < 0.001), and the incidences of SGA, preterm birth, PPROM, spontaneous preterm birth, and induced preterm birth were significantly higher in the subgroup with gestational weight gain below the optimal range (p < 0.001). In both groups, the frequency of cesarean delivery was lowest in the subgroup within the recommended weight gain.In the overweight group, the incidences of PIH and LGA were significantly higher with increasing gestational weight gain, and the incidence of macrosomia was significantly higher in the subgroup above the optimal weight gain (p < 0.001). In addition, the incidences of SGA, preterm birth, PPROM, and spontaneous preterm birth were significantly higher in the subgroup below the optimal weight gain (p < 0.001). The incidence of cesarean delivery was lowest in the subgroup within the recommended weight gain.In the obese group, the incidences of LGA, and macrosomia were significantly higher with increasing gestational weight gain (p < 0.001), and the incidences of SGA, preterm birth, PPROM and spontaneous preterm birth were significantly higher in the subgroup with gestational weight gain below the optimal range (p < 0.05); the incidence of PIH and cesarean delivery were not different by weight gain during pregnancy.The four pre-pregnancy BMI category groups demonstrated marked differences in the pregnancy outcome profile, indicating that the IOM-BMI classification is valid in Japanese women as well. In all groups, the gestational weight gain above the optimal range was associated with a higher incidence of LGA; the weight gain below the optimal range correlated with a higher incidence of SGA, preterm birth, PPROM, and spontaneous preterm birth; and the weight gain within the recommended range was associated with a better outcome. However, only the obese group did not show a difference in the incidence of PIH by gestational weight gain or a reduction in the incidence of cesarean delivery, even within the recommended weight gain.The BMI classification by the IOM was found to be valid in Japanese women.There were differences in the incidence of adverse pregnancy outcomes according to IOM recommended BMI categories. Due to a trend toward a higher proportion of leaner women and a lower proportion of obese individuals in the Japanese pregnant population, we defined those with BMI ≥25.0 as obese, in contrast to the IOM classification. However, the present study results indicated that Japanese pregnant women should also be categorized into an overweight group of pre-pregnant BMI 25–29.9 and an obese group with BMI ≥30. In addition, the group with BMI ≥30 did not show a difference in the incidence of PIH by weight control status during pregnancy or a reduction in cesarean delivery rate, even when the body weight was kept within the recommended range. These findings also indicated that weight reduction prior to pregnancy was important in improving pregnancy outcomes in obese women with BMI ≥30 in pregnancy. Poston et al.  reportedThe incidence rates of LGA and macrosomia were higher in women who had excess weight gain during pregnancy. Johnson et al.  reportedThe incidences of SGA, preterm birth, PPROM, and spontaneous preterm birth increased when the weight gain was below optimal. We previously reported that below optimal weight gain during pregnancy was associated with an increased risk of spontaneous preterm birth in underweight women ; howeverThe present study has limitations. First, our data set included only Japanese women, and it is unclear whether the results can be extrapolated to women of other ethnic groups. Second, we used the database of the JSOG Successive Pregnancy Birth Registry in tertiary centers. This may have introduced selective bias in the patient background characteristics. Third, we calculated the gestational weight gain as the change over the entire pregnancy period, and did not assess weekly weight gain.In Japan, pre-pregnancy BMI is currently classified into three categories: BMI <18.5, 18.5 to <25.0, and ≥25.0. On the basis of the present study results, we propose that the Japanese category of BMI ≥25.0 should also be further divided into overweight (BMI 25.0–29.9) and obese (BMI ≥30), in keeping with the IOM-recommended BMI classification. Our results also demonstrated that obese women with BMI ≥30 had a higher risk of adverse pregnancy outcome, and that gestational weight control did not have an impact on the cesarean delivery rate, supporting the importance of pre-pregnancy weight control within the normal range.


Background and Importance. Sellar arachnoid cysts and Rathke's cleft cysts are benign lesions that produce similar symptoms, including optochiasmatic compression, pituitary dysfunction, and headache. Studies have reported the use of various surgical treatment methods for treating these symptoms, preventing recurrence, and minimizing operative complications. However, the postoperative cerebrospinal fluid (CSF) fistula and recurrence rate remain significant. Clinical Presentation. In this paper, we present 8 consecutive cases involving arachnoid cysts and Rathke's cleft cysts, which were managed by using drainage and cisternostomy, the intentional fenestration of the cyst into the subarachnoid space, and then meticulously closing sellar floor using dural sutures. The postoperative images, CSF fistula rate, and the recurrence rate were favorable. Conclusion. We report this technique and discuss the benefit of this minimally invasive approach. The sellar nonneoplastic cystic lesions include Rathke's cleft cysts and arachnoid cysts , 2. The In this paper, we present 8 cases of Rathke's cleft cysts and arachnoid cysts, managed with endoscopically transsphenoidal surgery by applying intentional fenestration to the subarachnoid space and closing the sellar floor using delicate dural suturing technique. This method is minimally invasive and the surgical results were favorable.All patients were included in the prospective database between October 2009 and August 2013 who underwent endoscopic endonasal transsphenoidal surgery for treating symptomatic Rathke's cleft cysts and arachnoid cysts. The sample of 8 patients comprised 2 males and 6 females, with ages ranging from 37 to 73 years. In all of the subsequent patients, no packing of fat or other grafts was performed, wide fenestration of the cyst cavity to the SAS was intentionally conducted, and the sellar dura was closed meticulously using sutures and fibrin glue.Patient clinical notes, operative notes, imaging studies, and hormonal studies were reviewed. In addition, data on lesion characteristics, detailed intraoperative observations, intra- and postoperative complications, and clinical outcomes were collected. A single surgeon, Yudo Ishii, performed all of the procedures.Pituitary function was assessed using standard hormonal assays, including the levels of thyroid-stimulating hormones (TSHs) and thyroxine , growth hormones (GHs) and IGF-I, plasma adrenocorticotropic hormones (ACTHs) and serum cortisol, prolactin, luteinizing hormones (LHs), follicular-stimulating hormones (FSHs), and testosterone in men. In the early postoperative period, the patients were monitored for DI based on urine volume and urine-specific gravity. The hormone levels of the patients were monitored every 2 days after the operation.The preoperative and postoperative visual function assessment involved measuring visual acuity using formal visual field testing. Visual function was considered improved if the visual acuity assessed using the handheld eye card improved by at least 2 lines or if the visual field defects, assessed using field confrontation or by having an ophthalmologist conducting a formal visual field test review, were resolved or improved.All of the patients underwent pre- and postoperative pituitary MR imaging with and without Gadolinium enhancement, including early postoperative MR imaging on day 7 and subsequently within 3–6 months after operation. One of the female patients, the sixth patient, received a preoperative MR exam, but during the preoperative evaluation, a cardiac pacemaker was prescribed and, therefore, the postoperative image study was replaced with a brain CT scan.A direct endoscopic endonasal transsphenoidal surgery (ETSS) was performed in all of the cases in this study. The surgical procedure used for treating the RCCs and ACs in this study is summarized briefly, as follows.After performing a wide sphenoidotomy and sellar floor opening , the dur Staphylococcus epidermidis was cultured from the cyst content. He did not experience fever or meningitis postoperatively. But the patient received oral antibiotics for 4 weeks. The postoperative image in 3 months showed shrinkage of RCC. The RCC recurred in 6 months postoperatively. He received the same procedure with more thorough cyst content removal and fenestration into the subarachnoid space. The dura closure was achieved with suturing of his fascia lata. Three arachnoid cyst and 5 Rathke's cleft cyst cases were included in this series. All of the patient symptoms were visual disturbances. The patients did not exhibit headache or pituitary dysfunction, except for the fifth patient who had a headache, and the headache dissipated after the operation. The visual function of all of the patients was improved after the surgery. The preoperative MR image and the postoperation 3-4-month image are shown in An arachnoid cyst is a collection of CSF-like fluid, the walls of which comprise an arachnoid structure. The cyst can develop at any site in the subarachnoid space along the cerebrospinal axis. Two theories on the pathogenesis of intrasellar arachnoid cysts have been postulated . The firIntended fenestration of the cystic wall balances the pressure between the outside and inside of the cyst and can subsequently relieve the symptoms. The other way is that one of the pathogeneses of arachnoid cyst is the ball-valve mechanism. Once fenestration of the cyst with the subarachnoid space was performed, no pressure gradient occurred and, thus, the recurrence of arachnoid cyst was less likely. The wider the fenestration is, the faster the communication of subarachnoid space with the cyst is. The size of the fenestration was variable, depending on the relation between the small perforating arteries and the pituitary gland. We usually make fenestration until the flow of CSF becomes the to-and-fro oscillations of the arachnoid membrane like the 3rd ventriculostomy.We recommend that the fenestration site be located at the arachnoid membrane of the dorsum sellae, just above the posterior clinoid process. For Rathke's cleft cyst, the anterior pituitary gland is mostly located at the ventral sella, and the posterior lobe is at the dorsal sella. In endoscopic transsphenoidal surgery, we could verify the location of normal pituitary gland and the perforators supplying it. We make fenestration-avoiding injury to these vessels by direct vision under endoscope. In case of arachnoid cysts, pituitary gland would be located in the dorsal or caudal sella. In such cases, fenestration would be made at the frontal part of cyst as described by Oyama et al. Before the surgery, the surgeon should carefully examine the sagittal view of the MR image of the brain to determine the relationship of the trunk and tip of the basilar artery behind the dorsum sellae and possible location of pituitary stalk .Surgical therapy is the most commonly used method for treating symptomatic primary and recurrent Rathke's cleft cysts, and transsphenoidal approach is the preferred approach . SeveralConsequently, most neurosurgical centers limit the use of this complete resection method . The goaIn the treatment of Rathke's cleft cysts, fenestration of the cyst cavity to the subarachnoid space after removing the cyst content relieved the cyst pressure. The symptoms may thus be remedied. However, because the cyst wall was not completely removed, some residual ciliated epithelial cells may form the cyst content. The epithelial cell might be with a mucus secreting function, and the communication of subarachnoid space may prevent this secretion from accumulating. In the literature, studies have reported that “chemical meningitis” had occurred when removing the cystic craniopharyngioma . None ofThe postoperative recurrence rate and complication rate are high for these 2 diseases. The literature review and our result were summarized .CSF fistula is a major postoperative complication of endoscopic transsphenoidal surgery, especially in cystic lesions . Fat, geTherefore, we recommend not packing the cyst with fat or other materials after fenestration of the RCC with communication of the SAS. Although the flow of CSF may be large, the dura of the sellar floor can be closed using meticulously applied sutures and the synergistic easy slipknot approach, as reported by the senior researcher .We performed operations, totally 151 Rathke's cleft cysts and 5 arachnoid cysts from 2004 to 2013 in Nippon Medical School University Hospital with the method of traditional drainage and cyst wall biopsy. Among them, 10 Rathke's cleft cysts and 2 arachnoid cysts recurred. All the recurrent cases were large cysts with suprasellar extension. We propose the indication of this fenestration procedure for the large cysts with suprasellar extension. This approach would not be too invasive if it is performed in hands of an experienced endoscopic transsphenoidal surgeon with good sellar reconstruction technique, like the dura suturing. Endoscopic transsphenoidal management of the cystic lesions could be as easy as in craniotomy cases.Managing symptomatic RCC and sellar AC by fenestration of the cyst wall and meticulously applying dural sutures can provide symptom relief and prevent recurrence without increasing the risk of CSF fistula complications. Endoscopic endonasal transsphenoidal surgery to the cyst lesions can achieve more minimally invasive result than the extended approach method using microscope.The Supplementary Material is a demonstration video of this approach technique. This case is a recurrence of Rathke's cleft cyst. Endoscopically cyst drainage, cyst wall fenestration, dura suturing, and sellar reconstruction were demonstrated.


Plasmodium vivax infection on the balance of pro- versus anti- inflammatory cytokines and chemokines and their relationship with some clinical and epidemiology outcomes.  The study explored the effects of  Thirty-five pregnant women were recruited. Of these, 15 subjects had malaria at delivery (GM+), and 20 had no exposition to infection throughout the pregnancy (GM-) and at delivery. Epidemiological and clinical data were recorded after reviewing the clinical records. At delivery, whole blood from the mother as well as placental tissue was collected. Diagnosis of infection was performed by thick smear and a polymerase chain reaction (PCR). Expression of pro-inflammatory and anti-inflammatory cytokines and chemokines was measured by a real time PCR. The clinical and epidemiological variables explored were similar in both groups, with the exception of gestational age. When comparing the GM+ group with the GM- group, it is clear that although the differences generally are not significant, pro- inflammatory cytokines are elevated in both maternal blood and placental; anti-inflammatory ones are elevated in the mother and reduced in the placenta, and the chemokines are reduced in both compartments, except for MCP-1 which is elevated in all. P. vivax at childbirth. Additional studies are needed with larger groups in this and other regions of the country The results appear to be strongly affected by the small number of women with GM by  Plasmodium falciparum and very little is known about the role of P. vivaxP. falciparum as the main causing speciesPregnancy involves a greater risk of malariaPlasmodium infection in the mother changes the protective cytokines balance and increases the Th1 responses leading to complications such as anemia, abortion, stillbirth, low birth weight, among other. During a normal pregnancy the immune response undergoes specific changes to allow successful implanting and development of the fetus. Therefore, a Th2 profile is observed in successful pregnancies, while presence of a Th1 cytokine (pro- inflammatory) profile results in spontaneous abortion due to direct cytotoxic effects on the trophoblast. The normal anti-inflammatory status observed in the uninfected placenta is mediated by the increased production of cytokines, such as interleukin (IL)-10, and transforming growth factor β (TGFβ) which probably suppress the immune response mediated by cells. During GM-PM, this type of response is required to control symptoms, while the parasite is cleared by other mechanisms P. falciparum GM are tumor necrosis factor (TNF), gamma interferon (IFNγ) and IL- 10, but other cytokines and chemokines have also been exploredThe most studied cytokines in the physiopathology of , During placental infection, increased production of TNF and IFNγ results in injury of the villous trophoblastAccumulation of inflammatory cells, particularly macrophages and monocytes, in the inter-villous space, results after increased MIP-1β. Consequently, both the trophoblastic morphology and placental cytokine production are affected P. vivax, a species highly endemic in Asia and America. The aim of this research was to describe the expression profile of the pro-inflammatory cytokines TNF, IFNγ, IL-1β, the anti-inflammatory cytokines IL-10, IL-6, TGF-β and the chemokines IL-8 (CXCL8), MIP-1 β (CCL4) and MCP-1 (CCL2), in pregnant women and placentas infected with P. vivax at the time of delivery as well as to describe the association with clinical and epidemiological characteristics.Currently, no reports have been made on the immune balance during GM-PM by The study was carried out in north-west Colombia in the locality of Puerto Libertador (Cordoba department). Between Antioquia and Cordoba departments lays the malaria endemic region known as Uraba-Altos Sinu/San Jorge-Bajo Cauca, which has an estimated area of 43,506 km2 and a malaria at-risk population of 2.5 million distributed in 35 municipalities. P. vivax predominates over P. falciparum malaria in approximately a 2:1 ratioP. vivax, 34.4% of the cases of P. falcpiarum, and 45.7% of the cases by both especiesThis large region generates over 60% of malarial cases in Colombia: 18% in Córdoba and 42% in Antioquia. Subjects for the current study were recruited at the local hospital and the antenatal clinic of "CAMU Divino Niño", in Puerto Libertador.Volunteers were enrolled as part of a descriptive, prospective and transversal study, between June 2010 and June 2012. The sample size was defined by convenience and the lengthy period of recruitment was the result of severe and prolonged political turmoil in the region, which also jeopardized a random inclusion of subjects. Therefore, recruitment took place in a sequential fashion until the sample size for the study was reached.P. vivax GM at delivery (GM+); b) healthy pregnant women without GM during pregnancy and at childbirth (GM-). Recruitment took place regardless on the presence clinical signs of malaria infection at the time of diagnosis.Subjects were grouped as follows: a) parturient women with Thirty-five pregnant participants were enrolled: 15 with GM at delivery (GM+), and 20 without GM at the antenatal clinic or at delivery (GM-).P. falciparum infection.Inclusion criteria were>1 year residency in the study region, general good health and no history of serious disease, signature of the voluntary consent form, hospital delivery and attendance to at least three antenatal check-ups. Exclusion criteria were withdrawal of the voluntary consent and the finding of a PCR positive for Clinical data were collected after review of the antenatal clinical records and delivery charts. Information on mother´s age, weeks of gestation, parity, mother´s hemoglobin and newborn weight, was recorded.(r) preserved tissue fragment obtained from the placenta, were processed for expression of cytokines and chemokines.Whole blood samples from mother´s peripheral blood and placenta were obtained at delivery rooms in the local hospitals of Puerto Libertador. The mother`s sample was obtained by finger prick and placental blood was obtained from the lake formed following a wash with saline (0.9%) and removal of a 3x3x3 cm section in the area of the cord's insertion on the maternal side of the organ. These samples were used to perform thick and thin smear examination and PCR as detailed below. In addition, 4ml of maternal peripheral blood were collected and immediately stored in liquid nitrogen, this and a RNA laterPlasmodium vivax was confirmed in maternal and placental blood by thick smear and a nested polymerase chain reaction (PCR) according to standard published methods, The presence of (r) (Sigma(tm)). Amplification was carried out using a nested PCR assay to detect the 18 s rRNA gene of P. vivax, according to previously published proceduresP. vivax. Amplification products were resolved in a 2% agarose gel using ethidium bromide and visualized under UV light. Samples were processed once as long as controls were confirmed to operate in optimal conditions, otherwise, the assay was repeated until successful performance of controls.Diagnosis of infection by PCR was performed to whole blood samples collected onto Whatman 3MM filter paper, from which DNA was extracted using Chelex100For the GM+ group, at least one positive test was required for allocation and all tests were required negative for the GMC- group.The following definitions were applied: A) Low birth weight: <2,500 grams. B) Preterm delivery: a pregnancy concluded before 37 weeks. F) Maternal anaemia: haemoglobin <11 g/dL, G) Full term pregnancy: >38 weeks. H) Gestational or pregnancy malaria: plasmodial infection diagnosed in a pregnant woman after examination of maternal peripheral blood. I) Placental malaria: plasmodial infection diagnosed in placenta after examination of placental blood.(r) (QIAGEN) and cDNA was synthesized using First Strand cDNA Synthesis(r) (Fermentas). The quality of the cDNA was confirmed by using a conventional PCR for the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. The reaction was set up in an Applied Biosystems Step One Plus system using TaqMan(r). Results were read at 530 nm. the following genes were analyzed for expression: TNF, IFNγ, IL-1β , IL-10, IL-6, TGF-β, IL-8, MIP-1 and MCP-1 . The efficiency of the PCR reactions was determined based on mRNA extracted from a stimulated BeWo cell culture or peripheral mononuclear cells from a donor. cDNA was serially diluted and expression of β-actin was used to normalize the assays using the delta delta CT method (ΔΔCt). The reaction was performed in a final volume of 10 uL containing 1 uL DNA, 5ul TaqMan(r) master mix (Applied Biosystems), and 0.5 uL of 20X TaqMan assay mix (primers and probes for each cytokine/chemokine). All primers were supplied by Applied Biosystems. Reactions were performed at Laboratory of Tropical Diseases of the University of New Mexico, USA.Relative quantitation for expression analysis was performed using a reverse-transcription real time PCR assay (RT-PCR). Total RNA was extracted using QIAamp RNA Blood MiniData were analyzed using SPSS 10.0 . Proportions and medians were obtained. Significance was established at p<0.05%. The analysis included comparisons with using Mann-Whitney U test. Bivariate linear correlations were analyzed using Spearman´s rho coefficient.The study protocol was reviewed and approved by Ethics Committee of Instituto de Investigaciones Medicas, Universidad de Antioquia . Volunteers were recruited in accordance with national guidelines , as well as international guidelines , Edinburgh, Scotland, October 2000). Subjects were assigned a code which was known only by the researchers. A total 35 pregnant women were included in the study, 15 were allocated to the GM+ group and 20 to the GM-. . No significant differences were observed in the clinical and epidemiological variables between the two groups . In bothversus the GM- group; however, no significance could be confirmed in this result. Similarly, the chemokine MCP-1 was observed high in the GM+, but no significant difference was confirmed  was observed in the former. Significance in this finding was confirmed for TNF. Regarding the anti-inflammatory cytokines, these were lower in the GM+ group onfirmed .In maternal blood, all changes were not statistically significant. However, pro-inflammatory cytokines  were higher in the GM+ group with respect to the GM- group. In addition, all anti-inflammatory cytokines, but TGF-β, were increased in the GM+ group when compared to GM-. As for the chemokines, IL-8 and MIP-1 were lower, and MCP-1 higher, in the GM+ group when compared to GM-.P. vivax endemic region of America. The technique used to measure the expression of immune mediators is precise and sensitive. Volunteers were selected from the general population, particularly those regularly attending the hospital facilities in Puerto Libertador. Therefore, the study represents the typical population which seeks health services in the region.This reports for the first time on the status of cytokines and chemokines in women with GM in a An obvious limitation in this work is the reduced number of women with GM, which is the result of the current socio-political instability in the area. Despite the small sample size, the results obtained are consistent and suggest a clear trend. P. vivax infection using standard microscopy and contributes with the evidence, so far gathered, on the deleterious effects of this species The study contributes to outline the epidemiology of malarial infection during pregnancy in a highly endemic region of the country, since it includes subjects whose peripheral and placental blood was monitored for Other authors have reported on the particular predisposition to acquire malaria, regardless of the species, according to mother´s age, parity and gestational age P. vivax as responsible of low birth weight, maternal anemia and preterm delivery, Others have also reported on P. falciparum and this was associated with abortion, stillbirth, preterm delivery, intrauterine growth restriction, low birth weight and maternal anemia, P. vivax there is no report on this matter. We found an increased expression of all pro-inflammatory cytokines studied, both in maternal peripheral blood and in placental tissue. Although differences between the groups were not significant, the probability values observed strongly suggest that the sample size might have influenced this outcome. Similar to the findings herein reported, studies with P. falciparum GM also showed increased expression of TNF and IL1β but not IFNγ , . In other reports on P. falciparum GM, increased TNF has been associated with fetal growth restriction, spontaneous abortion and maternal anemia , Pro-inflammatory and anti-inflammatory cytokines and chemokines were found altered during GM by P. falciparum, P. vivax infected placentas. Moreover, a rise on IL-8 has been reported in placentas with active P. falciparum infection , The beta chemokine MCP-1 has also been reported to increase in GM and PM by P. vivax at the time of delivery. To consolidate these findings it is necessary to carry out additional studies with more women both in this region and elsewhere in the country.In summary, in women with GM at delivery pro-inflammatory cytokines tend to be elevated both in maternal blood and placental tissue, anti-inflammatory cytokines were high in the mother and low in the placenta and the chemokines were reduced in both compartments, but MCP-1 which was observed high in placenta and mother. The results seemed to be strongly affected by the relative small number of women with GM by


Adiponectin is an adipocyte hormone involved in energy homeostasis and metabolism. However, its role in early infancy is poorly understood.We recruited a total of 443 pregnant women and their children in this prospective study. Cord blood samples were successfully obtained from 331 neonates. Maternal and umbilical blood serum adiponectin were measured. The weight-, height- and BMI-for-age Z scores of infants at birth and at 3, 6 and 12 months of age were assessed.P < 0.01). Using Generalized Estimating Equation model after adjustment for sex, time, maternal age, gestational age, prepregnancy BMI, weight gain during pregnancy, maternal education, parity, history of miscarriage and mode of delivery, for every 1-μg/ml increment of maternal serum adiponectin, the height-for-age Z score during the first year of life increased by 0.026 (P =0.013) on average, and the height-for-age Z score of infants in the highest quartile of maternal serum adiponectin was 0.270 (95 % CI: 0.013–0.527) higher than those in the lowest quartile. The changes in weight-for-age Z score from birth decreased by 0.67 × 10−2 on average with every 1-μg/ml additional increase of cord blood adiponectin (P = 0.047). The infants in the highest quartile of cord blood adiponectin showed a −0.368  decrease in weight-for-age Z score change from birth compared with those in the lowest quartile.Multiple linear regression analysis indicated that cord blood but not maternal serum adiponectin was positively associated with all of the anthropometric measures at birth  contains supplementary material, which is available to authorized users. Obesity has received considerable attention as a major health threat with several etiologic influences . ObservaAdiponectin is an adipokine that is secreted by adipose tissue and correlates inversely with obesity in adults . It is aThe primary aim of this study was to determine whether maternal and cord blood adiponectin levels are related to the growth of children in early infancy in China.The subjects were 443 pregnancy women aged 20–35 and their babies from a prospective cohort study in Guangzhou, China. They were recruited from the Maternal and Children’s Hospital of BaiYun and Yuexiu District, Guangzhou between September 2010 and November 2011. All of the women had delivered normal single infants at full term. We excluded those with multiple births, a history of diabetes, chronic hypertension, endocrine disorders and /or other severe maternal illnesses. A detailed explanation of the study purposes, procedure and requirements was given to the subjects. Written informed consent was obtained from all the subjects before enrolment. The study protocol was approved by the research ethics board of Tongji Medical College, China.An interviewer-administered questionnaire was used to collect the demographic and historical information of the subjects, including their educational status, household income, personal medical and obstetrical history, information about their current pregnancy  and height.After delivery, maternal height was measured to the nearest 0.1 cm and weight to the nearest 0.5 kg while the subjects were wearing light clothing and no shoes. Measurements of the infants’ weight (±0.1 kg) and length (±0.1 cm) were made at birth and at 3, 6, and 12 months of age (+/− 15 days) according to standardised techniques by using an infant stadiometer (length board) and infant digital scale. Body mass index (BMI) was calculated as weight (kilogram)/height in meters square.Venous blood samples of mothers were obtained in the morning after overnight fasting when they were admitted to the hospital awaiting delivery. A total of 331 cord blood samples were successfully collected from the umbilical vein after the delivery of the baby and before the delivery of the placenta. All of the blood samples were placed on ice packs, stored in styrofoam containers, and returned to our laboratory. On arrival, the blood samples were centrifuged and aliquoted, and frozen at −80 °C until analysis. The adiponectin in the serum was determined by a commercially available ELISA . The intra- and interassay coefficients of variation were 5.4 and 7.0 %, respectively.P < 0.05 was considered significant. All continuous variables were expressed as mean ± standard deviation, or median with inter-quartile range. They were checked for normality using the Kolmogorov–Smirnoff test. Categorical variables are presented as percentages.All analyses were conducted using SPSS 21.0 for Windows . The age- and sex-specific weight-, height- and BMI-for age Z-scores were generated based on the WHO standards . Two mul2. The median increase in weight at the end of pregnancy was 15.0 kg. Fifty-two percent of the neonates were male. The median neonatal gestational age was 39.0 weeks. The adiponectin levels were significantly higher in the cord blood than in maternal serum . After adjustment for gestational age, weight gain during pregnancy and age, a weak positive correlation  was observed in adiponectin levels in maternal vs. fetal circulation. The anthropometric parameters at each visit are showed in Additional file Table -2 (P < 0.0001). Equivalent estimations of height- and BMI-for-age Z score at birth were 0.69 × 10-2 (P = 0.010) and 1.02 × 10-2 (P = 0.002), respectively. No significant relationships were detected between maternal serum adiponectin circulation and fetal anthropometrics.There were significant inverse associations between cord blood serum adiponectin level and the neonatal weight-, height- and BMI-for age Z scores Table . After c−2 on average during the follow-up period (P = 0.013). And the changes in weight-for-age Z score decreased by 0.67 × 10−2 with every 1-μg/ml increment of cord blood adiponectin (P = 0.047).The GEE analysis of the associations between maternal and cord blood adiponectin and the weight-, height- and BMI-for age Z scores and their changes at 3, 6 and 12 months are shown in Table When the maternal blood serum adiponectin levels were ranked in quartiles, the height-for-age Z score of infants in the highest quartile of maternal serum adiponectin was 0.270 (95 % CI: 0.013–0.527) higher than that in the lowest quartile  [r = 0.172, P = 0.004). Genetic variation in the adiponectin gene or its regulating elements may be responsible for this maternal-fetal correlation. In contrast, only nonstatistically significant positive correlations have been reported in most previous studies [n = 51–74) [We confirm data from previous observational studies showing that cord blood adiponectin levels were several folds higher than those seen in adults , 18. The 0.0001) . A signi studies , 20, 21.The positive correlation between cord blood adiponectin and fetal anthropometrics in our study is in agreement with some studies , 8, 21 br = −0.12; P = 0.05) and weight-for-age Z score  from birth to 6 months but was not associated with change in length-for-age Z score  [A few prospective studies have been conducted on the role of cord and maternal blood adiponectin levels in the postnatal growth of infants. In a sample of 588 children participating in the prospective prebirth cohort study Project Viva, Mantzoros et al. reported that cord blood adiponectin was inversely correlated with change in weight-for-length Z score (Most previous epidemiologic studies investigating the links between adiponectin levels and obesity in adults and children have demonstrated a negative relationship . In fetaThe association between maternal adiponectin concentration and fetal growth are less clear. A negative correlation with birth weight has been reported by some studies , but notThe strength of our study is its prospective design and repeated measures of anthropometric parameters in infants. We are the first to report the associations between adiponectin concentrations in maternal and umbilical blood and infant growth in early infancy in a Chinese population. However, our study has some limitations. First, not all of the subjects consented to the assessment of umbilical adiponectin status; thus, the results may be subject to potential bias. Second, the results may not be easy to generalize to other ethnic groups. Third, similar to all other previous studies, we did not determine fat mass directly, and did not measure the centrality of fat distribution. Thus, it remains unknown whether there is a relationship between adiponectin and visceral fat in infants. Finally, anthropometric measures during the follow-up had about 5.3–6.0 % missing data in our study. However, analyses after multiple imputation of the missing values yielded similar findings and thus our findings were not biased by the missing data.Cord blood adiponectin is a determinant of fetal size at birth and weight gain during the first year of life. Maternal serum adiponectin during pregnancy may predict postnatal height growth.BMI, body mass index; GEE, Generalized Estimating Equation; WHO, World Health Organization


Spatiotemporal hspb11 transgene expression in the musculature and the notochord matched closely that of endogenous hspb11 expression. Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression. Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency. The hspb11 transgene up-regulation induced by either chemicals or heat shock was eliminated after co-application of the anaesthetic MS-222. TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function.Robust and sensitive detection systems are a crucial asset for risk management of chemicals, which are produced in increasing number and diversity. To establish an  Danio rerio) were shown to be efficient for such toxicological estimation showing, for instance, a correlation and equal sensitivity to acute fish toxicity1The increasing diversity of chemicals produced by industry generates concerns regarding the potential hazards on biota and human health. The European Union’s REACH  regulation declares that the commercial producers are responsible for risk assessment and management of the chemicals that they produce. A major emphasis is placed on chemical safety requiring the evaluation of potential adverse toxicological outcomes and ecotoxicological hazards prior to selling the chemicals. Embryos of zebrafish  are known for their immediate responses upon exposure to a wide range of chemical stressors with little dependency on chemical structures3Motor dysfunction can be caused by problems in motor neurons and/or in the muscle. At the neuromuscular junction, a neurotransmitter, acetylcholine (ACh), is released from pre-synaptic terminals of motor neurons. Upon ACh binding to the post-synaptic nicotinic ACh receptor complex, the membrane of the muscle end plate depolarizes rapidly. This depolarization activates a cluster of voltage-gated sodium channels at the neuromuscular junctions and converts local end plate depolarization into a muscle fibre-wide action potentialTgBAC(hspb11:GFP) line specifically responded to chemicals that interfere with motor function and that cause induction of muscle hyperactivity. Dose-dependent up-regulation of hspb11 transgene expression was observed with a structurally wide range of chemicals and correlated with motility defects. We demonstrate that hspb11 transgene expression is induced specifically by muscle hyperactivity irrespective of the state of muscle integrity.We report here that the bacterial artificial chromosome (BAC) transgenic zebrafish hspb11 transcription was significantly induced by the pesticide, azinphosmethyl which impairs muscle integrityin vivo detection of chemicals causing motor dysfunction, we aimed at developing a transgenic zebrafish reporter line recapitulating the basal and induced expression patterns of hspb11. For this purpose, we obtained a BAC clone that includes the hspb11 locus and inserted a GFP reporter into the single exon locus of hspb11 by homologous recombination in E. coli22We previously reported that TgBAC(hspb11:GFP) transgenic line recapitulated the endogenous hspb11 expression pattern throughout embryonic development. At 24 hours post-fertilization (hpf), GFP-positive cells were detected in muscle pioneer cells at 24 hpf (hspb11 transgene is expressed in the slow muscles (hspb11 transgene is consistent with the endogenous hspb11 gene expression pattern (hspb11 gene (data not shown). Thus, we established a TgBAC(hspb11:GFP) line that faithfully reflects the endogenous gene expression with correct spatial and temporal distribution.The established  muscles ’, sm. Ro muscles ’. This e pattern . MoreoveTgBAC(hspb11:GFP) embryos with azinphosmethyl, propoxur and galanthamine, which exert their toxic effects on muscle through inhibition of Acetylcholine esterase (AChE) activityin situ hybridisation of azinphosmethyl treated and DMSO control embryos. An increase of gfp mRNA was detected in both slow and fast muscles in azinphosmethyl treated embryos but not solvent controls (data not shown).To test whether the transgene expression is responsive to compounds that cause muscle toxicity, we treated hspb11 transgene expression levels (p = 1.228 × 10−14). Consistent with a partial and not full recovery of muscle integrity assessed by birefringence, hspb11 transgene levels after the washout were higher than those of DMSO negative control for two of the three chemical treatments .To further ask whether the transgene expression level correlates with the reduced muscle integrity, we examined whether the transgene expression level would be reversed after recovery of muscle integrity by washing the compounds off. We compared GFP reporter levels between two groups for each of the three chemicals; one group treated with the chemical from 9–48 hpf, while another group treated from 9–24 hpf, followed by several washes in fresh fish water until 48 hpf . For allhspb11 transgene responds in a dose-dependent manner. We quantified the GFP reporter intensity in the trunk region (slow muscle + notochord) of TgBAC(hspb11:GFP) embryos after treatments with 14 test chemicals that have previously been shown to impair motor function. These chemicals exert their motor-toxic effects through inhibition of AChE, modulation of channel properties or other pathways (TgBAC(hspb11:GFP) embryos to these chemicals from 9–48 hpf and performed a semi-automatic quantitative assessment of GFP reporter intensityWe tested whether the pathways . All thepathways . This wapathways . We expohspb11 expression20hspb11 transgene expression was detected after exposure of embryos to the AChE inhibitors azinphosmethyl, propoxurhspb11 transgene expression in embryos exposed to this compound , of methylmercury that causes up-regulation of intracellular calcium levels, and of other channel modulators (methoxychlor and chlorophenol)  . For allmotility . Channelmotility . Thus, hhspb11 transgene response as an indicator of motor dysfunction, we also tested some other compounds with no previously reported effects on muscle birefringence in treated embryos: dibromoethane (oxidative phosphorylation disruptor), dimethylphenol (disruptor of ATPase activity) and chlorothalonil (thiol-reactive) (hspb11 transgene levels were not significantly different from solvent control after treatments with the three compounds (p > 0.001). Importantly, all three chemicals induced impaired motility  and chlorpyrifos (19.5-fold) further indicate the specificity of hspb11 transgene response  . The hspmotility , while nmotility . Given tmotility , the redresponse . Taken thspb11 up-regulation, we established a muscle specific Ca2+ biosensor line Tg([-505/-310]unc45b:GCaMP5A). We put the GCaMP5a cassetteunc45b gene , which confer skeletal muscle-specific reporter gene expression , propoxur (720 μM), galanthamine (1 mM) and veratridine (120 μM), with DMSO (0.05%) and dibromoethane (2.1 mM) as negative controls  test). Co-exposure with MS-222, however, abolished the up-regulation of hspb11 transgene expression by these compounds (p = 0.05185 one-way ANOVA), suggesting that muscle contraction is required for the up-regulation of the hspb11 transgene.For chemical induction of controls . In the hspb11 up-regulation and muscle disintegration when muscle contraction is absent. As reported previouslyhspb11 transgene with any of the treatments  showed normal birefringence  embryos at 48 hpf and measured hspb11 transgene levels at 4–5 hours after the heat shock.We observed that egration ’. Thus, ringence , comparahspb11 transgene levels at both 24 hpf (data not shown) and 48 hpf . In the presence of MS-222 during the 1-hour period of heat shock, however, up-regulation of hspb11 transgene expression was eliminated at both stages of development (p = 0.7098). This indicates that hspb11 transgene expression was up-regulated not by the heat itself, but rather by muscle hyperactivity during the heat shock. Accordingly, we observed increased motility during heat shock treatment in the absence of MS-222 (data not shown). Importantly, we did not observe muscle disintegration in the presence of MS-222 for 1-hour with or without heat shock; for each group n = 29/30 [97%] embryos showed normal birefringence. This result supports our hypothesis that it is not reduced muscle integrity but rather its hyperactivity that causes the up-regulation of hspb11 transgene expression.In the absence of MS-222, the heat shock treatment significantly increased TgBAC(hspb11:GFP) as a tool for monitoring chemicals that induce muscle hyperactivity. This transgenic line does not require time-lapse analysis for detecting muscle hyperactivity, as it converts a dynamic process into a quantifiable single readout. Because it does not assume any particular class of chemical compounds, it provides robust detection power. Indeed, chemicals that up-regulate hspb11 transgene expression show a wide variety of chemical structures  are expressed in the muscle, where they show induced expression upon heat shock31hspb11 transgene expression responds to muscle hyperactivity rather than directly to the heat shock raises the possibility that these “heat shock-inducible” sHSPs expressed in the muscle may also primarily respond to muscle hyperactivity. This unique hyperactivity-dependent hspb11 transgene induction precludes false-positive responses arising from general stresses associated with sample handling during a chemical screen. The high signal-to-noise ratio of up to 3-fold of GFP intensity after toxicant exposure is another beneficial feature of our hspb11-based system.One criticism against using heat shock proteins for any biosensor is the potential of a high false positive rate through their responsiveness to a wide range of stresses, such as heat shock or ROS exposure. However, our results show that the parallel . In zebrhspb11 transgene expression is regulated in a muscle activity-dependent manner, how should one interpret its expression in the notochord? Is our strategy to combine both muscular and notochordal hspb11 transgene expression inappropriate? To address this issue, we measured hspb11 transgene levels separately for both single tissues after chemical treatment (hspb11 transgene in both single tissues more than 1.5-fold over negative controls (DMSO and dibromoethane). Interestingly, the presence of MS-222 eliminated the up-regulation of hspb11 transgene both in the muscle and the notochord  zebrafish biosensor described here provides an unique tool for detecting chemicals that induce muscle hyperactivity, thereby interfering with motor function. It provides a quantitative measure for muscle hyperactivity without time lapse analysis, featuring high signal-to-noise ratio, sensitivity and specificity. A simple readout potentiates its use for increased throughput. Alternatively, the hspb11 sensor could easily be combined with the Ca2+ sensor by simply crossing differently coloured transgenic reporters into one line. Automated measurements of these fluorescent readouts combined with the measurements of altered behavioural effects such as the motor response .TgBAC(hspb11:GFP) and Tg(-[505–310]unc45b:GCaMP5A) were maintained on a 14 hr/10 hr light-dark cycle at 28.5 °C in a recirculation systems  and fed with commercial food and in-house hatched brine shrimp as describedet alWild-type zebrafish ABO is an inbred line initially derived from an intercross between the AB and wtOX line. The wild-type zebrafish strains ABO, and transgenic zebrafish lines hspb11 (DKEY-30K6 (ID:1132288); Sequence ID: AL844141.7; 257634 bp) was purchased from Source BioScience . The modified target vector with homologous arms of hspb11, enhanced green fluorescent protein (GFP) and kanamycin resistance sequences was generated as previously describedet al.Tg([-505/-310]unc45b:GCaMP5A), the 5′-untranslated region  of the gene coding for myosin chaperon unc45b was used to drive the calcium sensor GCaMP5A cassette in a muscle specific manner. The expression vector [−505/−310]unc45b-GCaMP5A flanked by Tol2-sites was created using the Tol2-kitThe zebrafish BAC clone for K6 ID:11388; SequeO,O-dimethyl S-[-yl)methyl]dithiophosphate; CAS# 86-50-0; dissolved in dimethylsulfoxide [DMSO]), propoxur , galanthamine – 5,6,9,10,11,12– hexahydro– 3-methoxy –11-methy l–4aH –ef] O,O-diethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate; CAS# 2921-88-2, dissolved in ethyl alcohol [EtOH]), chlorophenol , dibutylphthalate , veratridine -4,12,14,16,17,20-hexahydroxy-4,9-epoxycevan-3yl 3,4-dimethoxybenzoate 3-veratroylveracevine; CAS# 71-62-5, dissolved in DMSO), flucythrinate (cyano(3-phenoxyphenyl)methyl 2-[4-(difluoromethoxy)phenyl]-3-methylbutanoate; CAS# 70124-77-5, dissolved in EtOH), methoxychlor ethane; CAS# 72-43-5, dissolved in EtOH), esfenvalerate ((S)-cyano (3-phenoxyphenyl) methyl-(S)-4-chloro-alpha-(1-methylethyl); CAS# 66230-04-4, dissolved in EtOH), methylmercury (methylmercury(II) chloride; CAS# 115-09-3, dissolved in H2O), dibromoethane , dimethylphenol , chlorothalonil  and MS-222 .The chemicals used in this study were purchased from Sigma-Aldrich: azinphosmethyl  for preventing the formation of pigmentation. In dose response experiments for chemicals, embryos were treated in various concentrations between 0.01 μM to 3 mM. Final solvent (DMSO and EtOH) concentrations were never higher than 0.1%. Embryos were manually dechorionated at 48 hpf before imaging. The experiment was performed in three biological repeats with at least 10 embryos at each dose from a series of concentrations.Embryos were collected and raised in embryo mediumTgBAC(hspb11:GFP) transgenic embryos at 48 hpf were divided into 4 groups of 20–30 embryos each in the 2 ml tubes, filled with 1 ml of fish water. In two of the tubes the anaesthetic agent MS-222 was added at 643 μM, while the other two were kept as MS-222 negative. The temperature of the water bath was set to 37 °C and the heat shock was given for 1-hour to two groups of embryos, one with MS-222 and another without. The other pair of two groups, with or without MS-222, was kept at room temperature. After 4–5 hours post heat shock, images were taken under a compound microscope to quantify hspb11 transgene levels and to investigate the effects on muscle integrity by examining birefringence.For the heat shock experiment, The embryos were examined for motility and lethality at 48 hpf. Abnormal motility includes continuous twitch or shivering movements in the absence of touch stimuli, and lack of escape behaviour upon touch stimuli with the tip of a hair at the trunk. The experiment was performed in three biological repeats with at least 10 embryos each. Lethality was scored by lack of heart beat and coagulated bodies in a separate set of three experiments with at least 12 embryos each.Muscle integrity was observed under the compound microscope DM5000 with a 5 ×objective using polarized lightin situ hybridisation was performed as describedWhole-mount hspb11 reporter GFP signal was captured at a resolution of 1392 × 1040 pixels (px) with a physical spacing of 1 px = 0.32 μm. All acquired images were converted into a grey scale for further processing as described = 10 and region of interest extraction with a radius of r = 350 px). Global statistical values such as minimum, maximum, median, mean and standard deviation of intensity values were extracted from the linearized and cropped original image. All algorithms were implemented in MATLAB as an extension package to the open-source MATLAB toolbox Gait-CADhspb11 reporter level, the differences of the hspb11 reporter level among doses including solvent control were examined by one-way ANOVA with a significance threshold of p < 0.001. Chemicals with significant effects were modelled for their concentration dependent responses using a Gaussian 4-parameter logarithmic model after normalising the hspb11 transgene levels by the mean of the control . Subsequently, the concentrations (x) leading to a 1.5 (y) induction were calculated (EC1.5). The effect level of 1.5-fold was selected based on the average control variability. In case of methylmercury, dibutylphthalate, methoxychlor and 4-chlorophenol, no Gaussian like concentration-response was observed and the 1.5-fold induction levels were estimated from a linear regression modelThe coefficient Tg([-505/-310]unc45b:GCaMP5A) were embedded into 0.5% low-melting agarose prepared with 50 mM HEPES pH 7.0/ 5 mM NaCl/ 0.17 mM KCl/ 0.33 mM CaCl2/ 0.33 mM MgSO4. Each chemical treatment  was accompanied by solvent control (0.5% DMSO) in a home-made imaging chamber with two isolated slots. No anaesthetic chemical was added. A Leica TCS SP5 upright confocal microscope was used in resonant scanning mode  to achieve a time resolution of 2.5 minutes with a 2 μm z-step size for up to 12 embryos for 16 hours. A HCX PL APO lambda blue 20x NA0.70 IMM UV objective was used with glycerol as immersion medium. Temperature control was set to 28 °C. Data were processed with ImageJ/Fiji2-dpf transgenic embryos How to cite this article: Shahid, M. et al. Zebrafish biosensor for toxicant induced muscle hyperactivity. Sci. Rep.6, 23768; doi: 10.1038/srep23768 (2016).