Mucopolysaccharidosis (MPS) type III (Sanfilippo syndrome) comprises a group of rare, lysosomal storage diseases caused by the deficiency of one of four enzymes involved in the degradation of heparan sulfate. The clinical hallmark of the disease is severe neurological deterioration leading to dementia and death in the second decade of life. Adult MPS patients are generally of short stature. To date there is no clear description of the physical development of MPS III patients. The aim of this study was to document growth reference data for MPS III patients. We collected growth data of 182 German MPS III patients and were able to develop growth charts for this cohort. Growth curves for height, weight, head circumference, and body mass index were calculated and compared to German reference charts.Birth height, weight and head circumference were within the physiological ranges. Both genders were significantly taller than healthy children at 2 years of age, while only male patients were taller at the age of four. Growth velocity decelerated after the ages of 4.5 and 5 years for female and male patients, respectively. Both genders were significantly shorter than the reference group at the age of 17.5 years. Head circumference was larger compared to healthy matched controls within the first 2 years of life and remained enlarged until physical maturity.MPS III is a not yet treatable severe neuro-degenerative disease, developing new therapeutic strategies might change the course of the disease significantly. The present charts contribute to the understanding of the natural history of MPS III. Specific growth charts represent an important tool for families and physicians as the expected height at physical maturity can be estimated and therapeutic effects can be monitored.The online version of this article (10.1186/s13023-019-1065-x) contains supplementary material, which is available to authorized users. SGSH gene [Mucopolysaccharidosis type III  comprises a group of clinically indistinguishable, rare, autosomal recessive lysosomal storage diseases caused by the deficiency of one of four enzymes (defining the subtypes A-D) involved in the degradation of heparan sulfate (HS): heparan N-sulfatase (sulfamidase), α-N-acetylglucosaminidase (NAGLU), acetyl-coenzyme A α-glucosaminide-N-acetyltransferase and N-acetylglucosamine-6-sulfatase . The incGSH gene . PatientGSH gene . In addiGSH gene , 14, 15.GSH gene . There aGSH gene , 18. RecGSH gene .Disease-specific growth charts are important tools for tracking growth and recognizing deviations from normal. Physicians are able to counsel parents with regard to growth expectations. Even in a patient group where growth disturbance is not the severest pathology, growth charts might be important. Impact of possible new therapies for MPS III patients could change pathological growth to normal. Therefore, descriptions of the natural history of growth in MPS III patients are important to be able to assess changes.The aim of this study was to document growth reference data for MPS III patients and to describe the natural history of height, weight and head circumference in addition to growth spurts and clinical signs of puberty. We collected data of 182 German MPS III patients and developed charts for height, weight, head circumference and the body mass index.n = 7) were adjusted to the calculated date of birth.A retrospective chart review of 195 MPS III patients from two German specialized centers, was performed. Growth data of MPS III patients previously published by our workgroup were incorporated into the study . The diaSGSH gene.Twenty-five boys and 24 girls had already died at the time of data collection. The mean age at death was 16.8 years  for boys and 21.2 years  for girls. Overall 182 patients  were included in the study. These patients had a mean age of 17.7 years  in males and 19.1 year  in females at the time of assessment. Divided into subtypes, we had 135 MPS IIIA, 34 MPS IIIB and 11 MPS IIIC patients. Two patients had an unclassified subtype. Genetic data could be collected in 69 MPS IIIA patients. In this group we had 10 patients  carrying the mutation p.Ser298Pro on one allele of the Data from 1967 until 2015 were included. Gender, height, weight and head circumference (HC) were analyzed from birth until 21 years of age. Body mass index (BMI) was calculated from these data. Furthermore, data for age at thelarche, pubarche and menarche in girls and pubarche and growth of beard for boys were collected. Signs of puberty were retrospectively assessed by parent interviews. Patients over the age of 18 years were assumed to be fully grown. Data after the age of 18 years were integrated in the measurements for 18 year old patients.Our data were compared with the German KiGGS (Kinder- und Jugendgesundheits-Survey) reference percentiles for anthropometric measurements . As the p value < 0.05 was considered statistically significant.The statistical analysis was performed by using SPSS 20.0 for Windows, 22.0 for Macintosh  and Microsoft Excel 2010 for Windows . Mean height, weight, HC, and body mass index (BMI) were compared with a healthy German reference population [λ (power in the Box-Cox transformation), mu μ (Median) and sigma σ  LMS method [Growth curves for height, weight, head circumference, and BMI were calculated and plotted for each gender using the package gamlss (Version 4.3.3)  in R  and 51.1 cm (SD 2.8) for female newborns (n = 75). There was no significant difference to the reference group. At the age of 2 and 4 years male MPS III patients were significantly taller than the normal population . Female patients were significantly taller than the reference group at 2 years of age , but there was no significant difference at 4 years of age (104.5 cm versus 103.5 cm). Due to the limited availability of measurements between the ages of 5–17 years we were not able to obtain a statistically robust dataset for the comparison of height with the reference points. Descriptively, patients were shorter at age 5–7 years compared to the reference group. At 17.5 years of age both genders were significantly shorter than the reference group   4.64). The mean birth height was 52.3 cm (SD 2.88) for male . Data of thelarche was documented in 25/86 patients. Thelarche occurred at a mean age of 12.6 years . Signs of pubarche were documented in 51/96 male patients with a mean age of 12.6 years  and in 49/86 female patients with a mean of 13.9 years . Beard growth was seen in 17/33 patients . Excluded from analysis were two MPS IIIA patients  with central precocious puberty. The female patient had her pubarche at 6.75 and menarche at 6.5 years of age. Thelarche was seen with 7.5 years. The male patient was 7 years old at pubarche. Both patients were treated with gonatotropin-releasing hormone agonists.p < 0.000, mean reference group: 17.2 kg) and for female patients 18.7 kg . At age 18 male and female patients were significantly lighter   for male patients and 3.4 kg (SD 0.59) for female patients. At age 2 and 4 both genders had a significantly higher weight compared to the reference group. Weight for male patients at age 4 was in mean 20.0 kg  at 2 years of age and 17.0 kg/m2  at 4 years of age. The BMI of male patients at 2 years of age was 18.2 kg/m2 , at 4 years of age 17.8 kg/m2. BMI of male patients age 18 years  was significantly lower compared to the reference group (mean 22.3 kg/m2), but the sample size was small (n = 17). Female patients with 18 years of age showed no statistically significant difference to the reference group, but a tendency towards a lower BMI  , girls of age 2 had a HC similar to a 4.5 year old healthy female . At 4 years of age the HC was still significantly larger compared to healthy controls . Adult male patients had a HC in mean of 57.9 cm (SD 1.44), females in mean of 58.6 cm (SD 1.25)  at 2 years of age. Two years later they were still shorter in relation to other MPS IIIA patients. The final length of patients with the p.Ser298Pro mutation was higher than in patients without this mutation . MPS IIIA patients with the p.Ser298Pro mutation were slightly lighter at birth  compared to MPS IIIA patients with other mutations. At age 2 und 4 they were significantly lighter . Fully grown patients with the p.Ser298Pro mutation were significantly heavier . Head circumference was slightly decreased from birth up to age 4, but not significantly different to patients without the mutation. We had no data for HC in mature patients carrying the p.Ser298Pro mutation.Genetic data were available from 69 MPS IIIA patients. In 3 male and 7 female patients the p.Ser298Pro mutation was identified on one allele. There was no significant difference in growth between male and female patients with p.Ser298Pro. In comparison to MPS IIIA patients with other genetic mutations these patients were slightly smaller at birth  and significantly smaller .Newborn height, weight and HC were not significantly different to the reference charts. At the age of 2 years this changed. Affected boys and girls in this age group demonstrated a significantly higher body height, weight, BMI, and HC. The growth pattern of MPS III patients in our study was characterized by accelerated growth speed in the first year of life, with a subsequent deceleration and reduced height at maturity. These findings are similar to recently published data of a Dutch MPS III population. De Ruijter et al. described a normal birth weight and height, but showed a significantly stunted growth from the age of 6 years onwards . Adult hThe p.Ser298Pro genotype is present in about 10% of alleles in German patients with MPS IIIA. Patients with p.Ser298Pro genotype showed a more physiological growth with a higher body height at maturity. This reflects the milder clinical phenotype of patients with p.Ser298Pro genotype compared to classical MPS IIIA patients .Increased height at birth as well as accelerated growth in infancy has been reported for MPS I, MPS II, III, IVA, VI and VII . HoweverGrowth velocity has not yet been described in MPS III patients. In the present study multiple acceleration peaks were seen during growth in male MPS III patients. In contrast, female patients showed only one acceleration peak with 9 years of age. Clinical signs of puberty were not correlated to these growth spurts. Parini et al. also described a lack of pubertal growth spurt in MPS II . FurtherThe incidence of precocious puberty was 1.1%  in the present study, and therefore lower than reported in the literature. Concolino et al. observed precocious puberty in 2/10 MPS IIIA patients . Tylki-SThe pathophysiology of the short stature and the altered growth pattern in MPS III is not completely understood. Reports from the literature suggest a combination of several pathological mechanisms in bone formation, bone maturation, as well as endocrinological abnormalities –39. The It has been hypothesized, that GAG storage triggers a complex pathogenic cascade of abnormal biological mechanisms such as disruption of the extracellular matrix , alteratLimitations of this study are the retrospective design and the small number of data from 5 to 17 years of age , showing the construction of the charts with individual data points. (DOCX 142 kb)Additional file 2:Figure RD2. Raw data of weight (kg), showing the construction of the charts with individual data points. (DOCX 145 kb)Additional file 3:Figure RD3. Raw data of BMI (kg/m2), showing the construction of the charts with individual data points (DOCX 130 kb)Additional file 4:Figure RD4. Raw data of head circumference (cm), showing the construction of the charts with individual data points (DOCX 144 kb)Additional file 5:Table S1. Number of measurements for male and female patients for height, weight and head circumference in relation to the age groups (XLSX 11 kb)Additional file 6:Figure C1. Reconstructed charts for growth (cm) for MPS III patients, A: for boys (blue); B: for girls (orange). (DOCX 23 kb)Additional file 7:Figure C2. Reconstructed charts for weight (kg) for MPS III patients, A: for boys (blue); B: for girls (orange). (DOCX 23 kb)Additional file 8:Figure C3. Reconstructed charts for BMI (kg/m2) for MPS III patients, A: for boys (blue); B: for girls (orange). (DOCX 79 kb)Additional file 9:Figure C4. Reconstructed charts for head circumference (cm) for MPS III patients, A: for boys (blue); B: for girls (orange). (DOCX 21 kb)


Although the Chinese government has committed to reduce CH4 emissions under the requirement of the Copenhagen Accord (2009), there is lack of accurate CH4 emission data from young cows as the guideline of IPCC gives little consideration to the variations of geographic conditions, animal physiology stages, and dietary components of dairy production system. Our study investigated the effects of different dietary forage-to-concentrate on feed intake, growth performance, nutrient digestibility, and enteric CH4 emissions of Holstein heifers under various growth stage, and developed the prediction equations using production and emission data. Our results demonstrated that enteric CH4 emission was significantly affected by dietary composition and physiological condition; results obtained from the current study will be of great importance for development of regional or national emission inventories and mitigation approaches for heifers at specific growth stage.Enteric methane (CH4) emissions from young ruminants contribute to a substantial proportion of atmospheric CH4 accumulation. Development of emission inventory and mitigation approaches needs accurate estimation of individual emission from animals under various physiological conditions and production systems. This research investigated the effect of different dietary concentrate contents on feed intake, growth performance, nutrient digestibility and CH4 emissions of heifers at various stages, and also developed linear or non-linear prediction equations using data measured by sulphur hexafluoride tracer technique. Increasing dietary concentrate contents increased feed intake and growth rate, enhanced nutrient digestibility, and reduced enteric CH4 emissions. Heifers at the age of 9, 12, and 15 months with an average weight of 267.7, 342.1, and 418.6 kg produced 105.2, 137.4, and 209.4 g/day of CH4, and have an average value of CH4 energy per gross energy intake (Ym) 0.054, 0.064, 0.0667, respectively. Equations relating CH4 emission values with animal and feed characteristics were developed with high determination coefficients for heifers at different growth stages. Dietary concentrate contents had significant influence on overall performance of heifers. These data can be used to develop regional or national emission inventories and mitigation approaches for heifers under various production regimes in China.Enteric methane (CH As dietary structural carbohydrates  are degraded by ruminal microorganisms, CH4 emission represents up to 12% loss of dietary energy ingested in the rumen [4 emissions will help improve energy utilization efficiency and alleviate environmental pressures for dairy production regimes. According to the national report, CH4 emissions in 2005 from agriculture sector accounts for 25.5 Tg, of which approximately 57% is from rumen fermentation within ruminant production system in China [4 emissions in 2030 will reach 52.1 Tg [4 emission inventories [4 emission for the whole dairy population using a default value derived from lactating cows. In China, approximately 60% of the dairy population is milking cows, and the remainder is heifers  [4 emission of lactating cow and heifer, indicating the importance of quantifying the individual emissions for these animals [4 emissions of heifers under different physiological conditions. Therefore, the objective of the current study is to assess the effect of different dietary concentrate contents on the enteric CH4 emissions of Holstein heifers at various stage, and develop prediction equations using data collected using sulphur hexafluoride (SF6) tracer technique. Enteric methane CH is a finentories ,7. In adThis study was conducted in 2018 at the Zhongjiayonghong dairy farm located in Fangshan district, . Forty-five Chinese Holstein heifers with an initial body weight (BW) of 264.9 ± 25.6 kg (mean ± SD) were used in this study with three measurements taken at age of 9, 12, and 15 months, respectively. In each experiment stage, heifers were balanced by date of birth, age and BW and offered randomly assigned to 1 of 3 treatments (n = 15) in which animals were individually offered diets containing 30, 40, and 50% of concentrate . Each experimental period was 32 days in length, including 18 days for adaptation, followed by 8 days for gas measurement and 6 days for nutrient digestibility. Heifers were housed individually with free access to feed and water throughout the whole experiment. All animal care and handling procedures were reviewed and approved by the Animal Ethics Committee of Chinese Academy of Agricultural Sciences (protocol number 019–2018) prior to the start of the experiment. ad libitum . All diets in three periods were formulated to meet the recommendation of Ministry of Agriculture of P. R. China. For all of the three periods, cows were fed twice daily between 0600 and 0800 h, and 1600 and 1800 h. Feed refusals were collected and weighted to determine the daily feed intake.In period 1, cows received their diet as a total mixed rations (TMR) that composted of corn silage, wildrye, and a typical ration of concentrate on Chinese commercial farms. In period 2 and 3, alfalfa was included in the diet based on the ration of period 1 . The TMR4 emissions were measured from individual cows using the SF6 tracer technique with minor modification of Deighton et al. [6 by immersing in liquid nitrogen. The release rate was determined by incubating the permeation tubes in an oven at 39 °C and weighting each one twice a week for 4 weeks. The calculated releasing rate of the SF6 tubes ranged from 3.13 to 3.84  mg /day in period 1, from 3.10 to 3.70 (3.32 ± 0.266) mg/day in period 2, and from 3.10 to 3.60 (3.20 ± 0.167) mg/day in period 3, respectively. Each cow was randomly administrated with one SF6 tube using a balling gun three weeks before the commence of the experiment. Enteric CHn et al. . General6 and CH4 were also collected daily by using six additional canisters that were either placed on the back of animals or about 2.0 m above ground level of the experimental barn.A back-mounted harness was used to support the canister (volume = 1.85 L) for continuously sample collection; the canisters were washed by flushing 99.999% pure nitrogen and evacuated to over 98 kPa vacuum. The sampling rate of canister was approximately 0.25 mL/min by crimping a stainless-steel capillary tube within the sampling tubing. Canisters were removed after 24 h and residual vacuum was recorded before addition of nitrogen gas. Background gas samples of SF6 and CH4, respectively. Ultra-high purity nitrogen gas (99.999%) was used as carrier gas at 40 mL/min flow and analysis was performed after calibration with standard gases for SF6 and CH4. The daily CH4 emission was calculated as follows:4 is the calculated emission (g/d); SF6 is the measured releasing rate of each SF6 permeation tube (mg/day); the concentration of CH4 sample and CH4 background are expressed in ppm and concentration of SF6 sample and SF6 background in ppt; 6 and 146 are the molecular mass (g/mol) of CH4 and SF6, respectively; the factor of 1,000 is used to calculate CH4 in units of g/day. Gas samples were analyzed using a gas chromatography system  equipped with a flame-ionization detector (FID) and an electron-capture detector (ECD). The ECD operated at 300 °C with a molecular sieve 0.5 nm column and the FID at 150 °C with a Porapack N 80–100 mesh column  for determination of SFDuring the last 6 d of each experimental period (digestibility experiment), 5 cows out of each treatment were moved to metabolic stalls for nutrient digestibility measurement using a modified method of acid-insoluble ash (AIA) . GeneralRepresentative feed samples were collected during adaptation and experimental period for chemical composition determinations. Dietary gross energy (GE) content was determined by bomb calorimetry . Dry matter, neutral detergent fiber (NDF), acid detergent fiber (ADF), crude fat, and ash were determined using AOAC International (2006), and crude protein (CP) was measured using combustion analyzer .4 emissions was evaluated using two analytical approaches as described by Dong et al. [4 emissions. Prediction equations were developed using restricted maximum likelihood model as treatments were fitted as a fixed effect. Significant effects were noted at p < 0.05. The statistical program used in the current study was Genstat 14.2 .The effect of dietary concentration levels on growth performance, nutrient digestibility and enteric CHg et al. . GeneralDietary ingredients and chemical composition are presented in p < 0.05); however, these values did not differ significantly between the C40 and C50 treatments in period one or between C30 and C40 treatments in period two (p > 0.05). Dietary NDF intake was similar among the three treatments in period one , whereas heifers in C30 treatments consumed more NDF than the other two treatments in periods two and three (p < 0.05). Weight gain increased with increasing concentrate feed contents in the diet (p < 0.05) with an average ADG value of 1.26, 1.16, and 0.97 kg/day for heifers in periods one to three, respectively.Nutrients and energy intake and growth performance are presented in p < 0.05). However, DM and OM digestibility remained similar among the three treatments in period one, whereas both values increased linearly as concentrate increased in periods two and three (p < 0.05). Dietary ADF digestibility was unaffected by different concentrate contents in period two with a value of 68.4, 69.4 and 71.1 for C30, C40 and C50 treatments, respectively (p > 0.05). Moreover, there was an increasing trend in digestibility of average DM (74.47 vs. 75.50 vs. 78.47), OM (76.43 vs. 79.20 vs. 80.50) and ADF (68.42 vs. 69.63 vs. 74.00) for period one to three, respectively.Apparent nutrient digestibility data are presented in 4 emission data of each experiment period are presented in 4 production and CH4-E were significantly affected by treatments that both sets of parameters decreased linearly with increasing concentrate feed contents in the diets in any periods of one to three (p < 0.05). Enteric CH4 intensity including CH4/DM intake, CH4/OM intake, and CH4/NDF intake decreased linearly with increasing dietary concentrate feed contents throughout the three experiment periods, whereas no difference was observed for CH4/NDF intake between C40 and C50 (55.81 vs. 54.60 g/kg) treatments in period two (p > 0.05). CH4-E per gross energy intake (Ym) decreased significantly (p < 0.05) with increasing concentrate contents in any periods of one to three. Furthermore, although comparison of the effect of experimental periods on CH4 emissions was the objective of this study, the average of Ym value was 0.0564, 0.0639, and 0.0667 in periods one to three, respectively.Individual CH4 emissions in each period are presented in p < 0.01) with coefficient of determination values ranging from 0.27 to 0.74. Prediction equations of CH4 emission and DM intake in period three (Equation (8) in 2 = 0.74), whereas CH4 production was relatively poor related with BW for heifers in period 1 (Equation (1) in 2 = 0.47). Furthermore, emissions data derived from all three periods were pooled to develop overall CH4 prediction equations (4 emission (p < 0.01) and coefficient of determination value was 0.727 and 0.802 for linear and non-linear equations, respectively. Furthermore, a range of linear and non-linear prediction models were developed using the whole data sets of animal production and feed intake values (Equations (10) to (20), 2 can be observed with more variables were incorporated in to the models. For example, highest value of R2 of 0.820 was observed for the models relating CH4-E to BW, DM intake, and NFC intake (p < 0.01), whereas a relatively low value of R2 of 0.593 was observed for Equation (12), which relates CH4-E to DM intake and NDF intake (p < 0.01). However, there was no such trends for non-linear models as highest value of R2 was observed for the relationship between CH4-E and NDF intake (Equation (18)).Overall, relationships obtained in period three had highest values of determination values when compared with those from the other two periods. The strongest relationship was observed between CHquations . Feed inA number of studies have demonstrated that increasing dietary concentrate contents would increase feed intake of heifers, although they are typically fed high-fiber diets due to physiological and economic considerations . In accoGenerally, nutrient digestibility increased with increased concentrate supplementation for heifers in any periods of the present study. Moody et al.  reported4 emissions for cows at different ages and under various production systems. However, until recently, several studies investigated the effects of dietary concentrate contents on CH4 emissions for heifers. Boland et al. [4 emissions (121 vs. 132 g/day) for grazing beef heifer that consumed different herbage masses. Jiao et al. [4 emissions from heifer and steer at various growth stage, and reported an average daily CH4 emission of 93.5, 159.5, 175.0, and 188.5 g/day for young stock at the age of 6, 12, 18, and 22 months under confined condition. These values are similar to the recent study of Morrison et al. [4 emissions from grazing heifers using the SF6 tracer technique. However, emission data for heifers at the age of 15 months were higher than those for confined heifer and steer or for young stock in grazing condition [Due to the large population of China’s dairy industries, it is becoming increasingly important to quantify CHd et al.  reportedo et al.  examinedn et al.  who measondition . 4 emission represents the final production of ruminal fermentation via methanogenesis, it can be significantly affected by a range of factors including animal physiological state, dietary components, and measurement technique. Increased concentrate proportion resulted in reduced CH4 emissions in the current study. These values were consistent with studies of Muñoz et al. [4 production with increasing concentrate level up to 6 and 5 kg of concentrate per day, respectively. Generally, inclusion of high level of concentrate in the diet represents higher content of readily fermentable substance  than that of high forage diets. Previous studies demonstrated that starch-rich diets reduced ruminal pH and H2 concentration, and shifted fermentation patter towards to an increased propionate formation, which would depress the activity of methanogens and consequently reduce CH4 emissions [4 emissions of heifers [4 emissions as the growth of heifer advanced, which would reflect the changes and distribution of ruminal methanogens. As enteric CHz et al. , who alsmissions ,31. More heifers . Althoug4 emission inventories in many countries are currently estimated using the Tier-2 methodology from International Panel on Climate Change (IPCC) guidelines. As with Tier-2 approaches, default prediction values for Ym from adult dairy cows in the 1997 (0.060) [4 estimation of the whole dairy population. However, adoption of a default and fixed value has becoming a major concern because it can vary considerably with varying geographic conditions, cow breed and physiological stages, and dietary characteristics [m value of 0.067 or 0.076 for yearling heifers either fed ad-libitum or under restricted feeding condition. Morrison et al. [m values for calves, yearling heifer, and in-calf heifer with an average age of 8.5, 14.5 and 20.5 months, and found that the calculated Ym was 0.057, 0.0675, 0.059 for each period. In accordance with those results from confined lactating cows or heifers at pasture, a range of Ym values between 0.0454 and 0.0769 were obtained across all heifer ages in the current study, which lied within the range (0.036–0.114) obtained under diverse production systems [m values were also examined when heifers were fed different concentrate contents in the diet. As report previously, variations of dietary components such as starch: NDF ratios can change the rumen fermentation environment and methanogenesis functions, which consequently affect the CH4 emissions. In the present study, Ym values decreased from 0.0686 to 0.0454, 0.0742 to 0.0558, 0.0769 to 0.0568 when concentrate intake increased from 1.68 to 3.45, 2.09 to 3.59, 2.23 to 3.98 kg/day in period 1 to 3, respectively. These results are consistent with the grazing studies of van Wyngaard et al. [m of lactating Jersey cows significantly decreased from 0.0891 to 0.0785 when the concentrate increased from 0 to 8 kg/day.Regional or national enteric CH 0.060)  and 2006 (0.060)  IPCC gui0  and 20eristics  reportedn et al.  calculat systems . Therefod et al. , who rep4 emission predictions have been widely developed based on mathematical or statistical association of nutrient intake, dietary nutrient composition and digestibility and other animal factors with enteric CH4 emissions [4 emissions in this study with values of R2 ranging from 0.67 to 0.74. Similar R2 values of 0.68 with DM intake and 0.70 with GE intake were reported for beef cattle measured using respiration calorimeters [4 emissions, and suggested that DM intake alone may be sufficient to achieve satisfactory prediction accuracy inventory purposes [4 emissions, with a high value of determination coefficient and an intercept close to zero when DM intake ranged from 2 to 28 kg/day. The data was pooled together and a linear or nonlinear relationship was observed between BW, DM intake and CH4 emissions in the current study. However, curvilinear relationship between DM intake and CH4 production was observed when dairy cows were fed relatively high proportion of concentrate [4 production can be achieved when the concentrate level was below 30% [4 emissions were highly affected by several factors including growth stage, dietary concentrations of protein and carbohydrate fractions. For example, coefficients for DM intake increased from 24.21 to 51.72 for heifers at the age of 9 to 15 months, although this difference did not reach significance. Moreover, lower values of R2 from 0.42 to 0.47 were observed when animal characteristic such as BW was used as a single predictor variable. Jiao et al. [4 for an increase of 1 kg of heifer BW, which was similar to our findings that an average increase of 0.203 kg/day CH4 was observed for each unit increase of BW.Enteric CHmissions . In agrerimeters . Appuhampurposes . A meta-purposes  showed tcentrate . It was elow 30% . Yan et elow 30%  reportedo et al.  reported4 emissions may not follow a linear trend as generation of CH4 can be affected by ruminal function and fermentation dynamics. Among the non-linear models developed using the whole data sets of the present study, a highest R2 value of 0.82 was observed when BW, DM intake and NFC intake were incorporate in to the equation. However, a range of relative lower values of R2 were also found for those exponential or power equations. This result was in consistent with the previous research of Mills et al. [4 production in a wide range of production variables; especially, as they could be more appropriate when extreme values were obtained during the practical application [4 emissions, whereas increasing dietary NFC intake may reduce CH4 emission. Diets rich in non-structural carbohydrates such as starch and sugars are converted to propionate in the rumen with less hydrogen and CH4 production. However, fermentation of fibrous materials would favour the formation of acetate and butyrate, which would have positive impact on CH4 emissions. Although linear models can be mathematically developed using dietary intake and composition variables, enteric CHs et al.  and Patrs et al. , who fous et al.  suggestelication . The slo4 conversion factors are derived from the current study, reflecting the variations of animal and dietary characteristics under the typical production regimes in China. Together with prediction equations, these data will be of great importance for development of regional or national emission inventories and mitigation approaches for heifers at specific growth stage.It is concluded that increasing dietary concentrate contents improves feed intake and growth performance, and nutrient digestibility. Enteric methane emissions decrease significantly with increasing concentrate contents. A range of CH


Schistosoma mansoni prevalence in the seven MDA districts decreased to 20.4% (95% CI: 18.7–22.3%) in 2016 from 42.2% (95% CI: 39.8–44.5%) at baseline (p < 0.0001). Mean overall S. mansoni intensity of infection reduced to 52.8 epg (95% CI: 43.2–62.4 epg) in 2016 from 100.5 epg (95% CI: 88.7–112.3 epg) at baseline (p < 0.001). The prevalence of Schistosoma haematobium in the five MDA districts that had baseline prevalence data decreased to 2.2% (95% CI: 1.5–3.1%) in 2016 from 18.3% (95% CI: 16.3–20.5%) at baseline (p < 0.0001). Mean overall intensity of infection increased to 1.12 e/10 ml (95% CI: 0.55–0.1.70 e/10 ml) in 2016 compared to 0.47 e/10 ml (95% CI: 0.16–0.78 e/10 ml) in 2012 (p < 0.05) (no baseline data). No district was highly endemic in 2016 compared to three at baseline and there was no significant difference in prevalence or intensity of infection by sex for both species. This survey illustrated the significant progress made in controlling schistosomiasis in Sierra Leone. The fact that prevalence and intensity of infection showed an increase from the 2010 level suggested a detrimental effect of missing MDA due to the Ebola toward schistosomiasis control. The national program needs to continue the treatment and adopt a comprehensive approach including water, hygiene, and sanitation measures to achieve control and elimination of schistosomiasis.Historic data and baseline surveys showed schistosomiasis as highly/moderately endemic in 7 of 14 districts in Sierra Leone, justifying annual/biennial mass drug administration (MDA) with praziquantel. MDA commenced in 2009 and reported treatment coverage had been above the World Health Organization recommended 75% of target population. Assessment in 2012 showed significant reduction in infection. In 2016, another national school-based survey was conducted to evaluate the progress. Two schools from each category  of endemic communities in each MDA district and five schools in non-MDA districts were selected. Fifty children (25 boys and 25 girls) aged 9–14 years were randomly selected per school. Parasitological examination of 1,980 stool and 1,382 urine samples were conducted. Overall  Schistosoma mansoni  and Schistosoma haematobium  that are transmitted via intermediate host snails Biomphalaria sp. and Bulinus sp., respectively  worldwide  and secoectively –7.Chronic intestinal schistosomiasis is manifested by hepatosplenomegaly , while uSchistosomiasis is one of five NTDs targeted with preventive chemotherapy for control/elimination. The World Health Organization (WHO)  strategyS. mansoni and S. haematobium were endemic in Sierra Leone  coastal districts  and at risk adults in all seven highly/moderately endemic districts in 2010. It was thought that transmission was not possible in the brackish waters of the coastal districts and that the few infected individuals who were found during baseline surveys were displaced from inland districts during the war and therefore MDA has not been conducted in these coastal districts to date.S. mansoni infections , then social mobilization at community level. At-risk adults and non-enrolled SAC were targeted by community-based MDA and enrolled SAC were reached at school-based MDA. Owing to the potential adverse events of PZQ in heavily parasitized individuals the national policy has been that health workers (rather than volunteers or teachers) should be the distributors and that PZQ should only be given after a meal . TreatmeIndependent monitoring of MDA coverage was conducted within a week of the MDA to assess performance and to validate the reported coverage, using the methodology described previously . In 2015The details of the baseline mapping surveys were described previously , 30. BriIn 2016, a cross-sectional school-based survey was conducted in all 14 districts 6 months after the last MDA. A total of 72 sentinel sites were surveyed. Of these, 40 sites (schools) were from low, moderate and high prevalence communities identified at baseline from each of the seven MDA-districts, 5–6 sites per district, and additional 32 sites were from the seven non-MDA districts, 4–5 sites per district. To avoid repeated sampling that may raise awareness and bias the results, 38 sentinel sites were retained from those surveyed either at the baseline or in 2012 and 34 new sites were selected in 2016 [22 WHO]. Approximately 50 children aged 9–14 years were randomly selected per school.Nationwide up to 38% of households were still reliant on an unimproved water source for drinking, of which 31% fetch water from a river or stream. In 10 districts, less than 10 percent of households had access to improved sanitation .Upon arrival of the survey team at school, children were randomly selected according to the calculated sampling intervals and given a unique identification number, and their sex, and age were recorded. They were each given two plastic containers and asked to provide a fresh stool sample and a midday (10 a.m.−2 p.m.) terminal urine sample. Prior to the urine collection, about 15 min exercise was initiated by the survey team and teachers. All stool samples were examined (1 slide per sample) within 5 h of collection by the Kato-Katz method using a 41.7 mg thick fecal smear template and microscopy with a x10 lens . Number In 2008, Kato Katz was used for fresh stool examination in the field, but intensity was not calculated. In 2009, the stool samples were preserved using 10% formalin and transported to a laboratory in Freetown for examination. Fresh terminal urine samples were examined in field following manual centrifugation for 5 min and intensity data was not calculated.S. mansoni was categorized as heavy (≥400 epg), moderate (100–399 epg), or light (1–99 epg) and for S. haematobium as heavy (≥50 e10 ml) or light (<50 e10 ml). GPS coordinates of the sites were collected and QGIS, Version 2.18.15 used to plot point prevalence distribution.Data were entered into Microsoft excel spreadsheet and exported for analysis using SPSS statistical software . The SPSS Complex Samples Crosstabs was used to calculate the prevalence and the Complex Samples Descriptive to calculate the mean egg counts with 95% confidence intervals (CI), taking into consideration the clustered nature of school children using districts as strata and schools as clusters. The arithmetic mean egg counts of infection from all those examined (including both positive and negative) was used in the analysis. Chi-squared test was used to compare prevalence and the Kruskal-Wallis test was used to compare mean egg counts between the studies and by sex. Intensity for The surveys were part of the monitoring and evaluation activities of the national NTDP. Ethical approval for the survey was obtained from the Ethics and Scientific Review Committee of the Ministry of Health and Sanitation (MoHS), Sierra Leone. Community informed consent was obtained following discussion with District Medical Officers, head teachers, and community leaders. All communities were sensitized prior to sample collection and verbal consent was sought from parents. Written consent was obtained from the head teacher on behalf of the selected pupils who were also sensitized on the purpose of the activity. All children participated voluntarily, and the names were anonymized following sample collection. Those found positive were referred to the following MDA with PZQ.S. mansoni was 11.4% (95% CI: 10.4–12.5%). It varied significantly from 0 to 1.6% in the seven non-MDA districts and 2.7–39.5% in the seven MDA districts. Kenema, Koinadugu, and Kailahun were among the districts with highest prevalence . Overall in all 14 districts (both MDA and non-MDA) there was no statistically significant difference in prevalence by sex: males 11.5% (95% CI: 10.1–13.0%) vs. females 11.3% (CI: 9.9–12.8%) (p > 0.05). This was similar when using data from the seven MDA districts: males 20.6% (95% CI: 18.2–23.3%) vs. females 20.2% (CI: 17.7–22.8%) (p > 0.05) as shown in In 2016, 1,980 stool samples from school age children  and 1,382 urine samples  were examined from 14 districts. The results are summarized in S. mansoni prevalence in the seven MDA districts that were highly/moderately endemic, there was a significant reduction in overall prevalence from 42.2% (95% CI: 39.8–44.5%) at baseline to 20.4% (95% CI: 18.7–22.3%) in 2016 which was however a slight increase from 14.9% (95% CI: 13.2–16.7%) in 2012 (p < 0.001) , with Kambia, Pujehun, and RWA showing even lower prevalence than the baseline (p < 0.01)  . In the  < 0.01) .p > 0.05); the other six districts showed significant decrease in 2012 from the baseline (p < 0.01); but in 2016 Kailahun, Kenema, and Koinadugu showed significant rebound in prevalence from the 2012 level (p < 0.01) while Bo and Tonkolili stayed flat from 2012 and only Kono continued to decrease (p < 0.05 each) as shown in Among the seven MDA districts, Bombali did not show significant change in prevalence over three surveys (decrease . Both map > 0.05). The few infections in the seven non-MDA districts were very light and no comparison was made due to the lack of egg count data at the baseline.The overall mean egg count in 2016 was 29.1 epg (95% CI: 23.8–34.4 epg) in 14 districts as shown in p = 0.001) (p < 0.05 each). Overall there was a significant increase in 2016 from 18.4 epg (95% CI:14.7–22.1 epg) in 2012 (p < 0.001) (p = 0.001 each) from the baseline, but a significant increase from 2012 (p < 0.01).Compared with the baseline (2009), among the seven MDA districts, there was a significant reduction in overall mean egg count from 100.5 epg (95% CI: 88.7–112.3 epg) to 52.8 epg (95% CI: 43.2–62.4 epg) in 2016 (= 0.001) . Four of< 0.001) . Four di< 0.001) . Only Kop < 0.0001), but significantly increased from 1.1 and 1.0% in 2012 (p < 0.001)  of heavy infections across the 14 districts and 5 districts  had heavy infections between 2.7 and 10.0% . Among t< 0.001) .S. haematobium infection with an overall prevalence of 1.8% (95% CI: 1.4–2.3%), but mainly in the seven MDA districts  heavy infection, with only two districts above 1% (Koinadugu 2.0% and Tonkolili 1.3%).There was a low level of istricts . Among tS. haematobium prevalence from 18.3 (95% CI: 16.3–20.5%) at baseline to 2.2% (95% CI: 1.5–3.1%) in 2016 (p < 0.001) as shown in p < 0.05). No district had moderate or high prevalence in 2016 compared to 2 districts (Bo and Kono) with moderate prevalence at baseline. The mean intensity was 1.1 e10 ml (95% CI: 0.6–1.7 e10 ml) in 2016 (no baseline intensity data available).Only five districts had baseline data and therefore the data from these five districts were used for comparison between the surveys. There was a significant reduction in overall p < 0.005). The overall mean intensity increased from 0.5 eggs/10 ml (95% CI: 0.1–0.9) in 2012 to 1.1 eggs/10 ml (95% CI: 0.6–1.7) in 2016 (p < 0.05).Overall, or by sex there was no significant change in prevalence compared with 2012. One district (Bo) showed significant reduction in prevalence  to treat at least 80% of SAC and at-risk adult population in endemic communities with PZQ and (2) to reduce prevalence among SAC in all districts to less than 10% . ReporteS. mansoni from the baseline. For S. haematobium in the five districts that had baseline data there was an 88.0% reduction in prevalence. Two of the seven MDA districts reached the national target of reducing prevalence in SACs to below 10%, while the other five MDA districts still had 13.9–39.5% S. mansoni prevalence with heavy infections at 2.7–10.0% after six rounds of MDA over 7 years.In line with the mapping results –32, the Despite the success achieved in the overall MDA coverage, there had been challenges in reaching some people. In certain places, the actual coverage may not have been as high as reported. The post-event surveys by independent monitors found MDA coverage had been lower than that reported by the NTDP especially in two districts (Bo and Kailahun). Possible explanations may include: (1) underestimates of target populations as the projected growth rate of 2.4% in the national census in 2004 was amended to 3.2% in the national census in 2015; (2) the biennial phases of MDA in chiefdoms in Bo where SAC move considerably to attend educational resources away from home; (3) in Kailahun which borders both Guinea and Liberia the considerable cross-border migration to access PZQ during MDA in Sierra Leone; and (4) limited resources for health workers to return to communities for missed eligible persons in remote communities. Independent monitoring also identified common reasons for noncompliance in taking PZQ which included experience of previous adverse events either by self, a close friend or relative such as nausea, dizziness, diarrhea, vomiting, and headache.S. haematobium infection in Tonkolili could be attributed to the rapid employment-seeking migration from more highly endemic communities as a result of the opening a railway serving new iron-ore mines in 2012 and/or alterations to snail populations from irrigation systems built to sustain a large sugar cane plantation and a dam for hydroelectricity production. In addition, ~50% of the sentinel sites from the baseline were changed in the 2016 survey, with purposefully selected sites from other areas where no baseline data were available. All these may have contributed to the increases in both prevalence and intensity of S. mansoni seen in this survey in 2016 from 2012.Prevalence and intensity in 2016 did not show further reduction after 2012 and in three border districts there was an increase: Kailahun, Kenema, and Koinadugu. One of the factors may have been due to the fact that only one round of MDA occurred between 2012 and 2015 in moderately endemic communities and heavily infected communities also missed one round of MDA due to the Ebola emergency in 2014. In Kenema which had the highest prevalence in 2016, one of three original sentinel sites (Lower Bambara) had the highest prevalence and intensity at baseline , suggesting a high level of local transmission in certain areas. The increase at one site in Port Loko and of S. mansoni from baseline (S. haematobium remained high (>18%) in 3 of the 11 MDA-regions  and sanitation . In 2015, an estimated 50% of rural households were still reliant on unimproved sources  for drinIn line with the WHO recommendations  and baseThere were certain limitations in the study. Firstly, there were differences in the sample sizes and sampling methods used in different surveys in different years. This made the comparison of the results between years difficult. Secondly, the surveys were conducted in school age children, and the situation in other at-risk age groups is not known. Future surveys should consider sampling from other at-risk age groups. Other potential cofounding factors that may have influenced these results relate to personal hygiene, access to safe drinking water and improved sanitation. There has been considerable effort to improve the practice of hand-washing with soap and the safe disposal of human feces before which was intensified during the Ebola emergency but overall the situation remains poor and continues to need attention if transmission is to be reduced and improvements sustained.In conclusion, by 2016 Sierra Leone had made major progress in reducing prevalence and intensity of schistosomiasis infection, compared with the baseline in 2008/9. However, the Ebola outbreak in 2014 and other factors have hindered the progress of schistosomiasis control. The NTDP should continue MDAs and endevour to reach all at risk communities especially in the three border districts whilst coordinating with WASH partners and their activities to reduce transmission.Although consent was obtained from participants before the study, data collection was conducted such that participants remained anonymous during data entry and analysis. No individuals' identity can be revealed upon publication.All data generated or analyzed during this study are included in this published article. The dataset analyzed is available from the corresponding author on reasonable request and can be made available with permission from the MoHS Sierra Leone.The impact assessment was part of the routine monitoring and evaluation activities of the national NTDP. Ethical approval was obtained from the Ethics and Scientific Review Committee of the MoHS. Community informed consent was obtained following discussion with District Medical Officers, Chiefdom school inspectors, head teachers and community-teachers' associations. All communities included in the survey were sensitized by representatives of the district NTDP and the survey teams upon arrival prior to sample collection. Verbal consent was sought from parents during these meetings. A written consent was obtained from the school head teacher and the village head on behalf of the pupils recruited. Children enlisted were also sensitized on the purpose of the activity prior to sample collection. Participation was voluntary. All data generated or analyzed during this study are included in this published article and its supplementary files.YB and AC managed and supervised the national NTDP. JP, MB, MS, AV, and MH planned and coordinated the survey. JP, MB, AT, and SS collected the field data and performed the laboratory investigations. MB and JP performed the data analysis. JP wrote the first draft of the manuscript. MH and YZ revised the paper. All authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CYP1A1 which belong to cytochrome P450 (CYPs) superfamily, is a very important gene for the metabolism of carcinogens.Endometriosis is a disease that affects women of reproductive age. This disease is characterized by the presence of endometrial-like tissues  outside the uterus and shows significantly elevated prevalence in industrial regions. Additionally, an interaction between genetics and environmental factors is assumed for the disease. Enzymes belonging to the cytochrome P450 (CYP) family are participated in detoxiﬁcation process of a wide range of environmental toxins and carcinogens. Thereby, they are good link for the interaction. MspI polymorphism of CYP1A1 gene and its relation to endometriosis.The aim of this study was to analyze the frequency of the Genomic DNA was isolated from 93 endometriosis women and 139 healthy controls. Genotyping was performed using polymerase chain reaction followed by restriction fragment length polymorphism analysis. CYP1A1 gene polymorphism in patients were 73.1%, 22.6%, and 4.3%, while frequencies in controls were 74.1%, 22.3%, and 3.6%, respectively. So there was no significant differences between the genotypes in two groups (p=0.961). Frequencies of the TT, TC, and CC genotype of MspI polymorphism of CYP1A1 gene appears to be not associated with the risk of endometriosis in the studied population. However, additional studies, especially with larger sample size are needed to validate these findings.According to our study,  CYP) P450 superfamily are the most important ones and possibly a good link for interactions of genetic and environmental factors  (CYP1A1 rs4646903 (T>C) polymorphism, downstream from the polyadenylation site at the 3´ end of exon 7, is one of the most important polymorphisms of the gene. Substitution of T to C at this position formed a target site for MspI enzyme, so this polymorphism is also named MspI or M1 (MspI polymorphism and the risk of endometriosis (5q 24.1) . CYP1A1 pI or M1 , 12. It etriosis , 14. MspI polymorphism of CYP1A1 gene and endometriosis in women referred to two infertility centers in Yazd, Iran who were from central and southern parts of Iran.The goal of this study was investigation of the association between In this case-control study, 93 women with endometriosis (age range 20-42 yr) referred to two infertility centers in Yazd, Iran: Madar Hospital and Yazd Research and Clinical Center for Infertility were enrolled in this study as the case group. Endometriosis was diagnosed by an experienced gynecologist through laparoscopy. Guidelines of American Society for Reproductive Medicine (ASRM) was used to classify patients (ASRM) into 4 stages; minimal (stage I), mild (stage II), moderate (stage III), and severe (stage IV) . The control group consisted of 139 unrelated healthy premenopausal women (age range: 18-50 yr) undergoing Cesarean section or hysterectomy at the same centers with no history of endometriosis and without any lesion suggesting endometriosis during Cesarean section or hysterectomy. Women with a history of autoimmune disease and cancer were excluded from the research. Clinical characteristics of all the participants were collected personally using a designed questionnaire. Genotype determinationMspI polymorphism of CYP1A1 gene, PCR-based restriction fragment length polymorphism (RFLP) method was used. The specific primers for the target region (forward primer of 5'-TAGGAGTCTTGTCTCATGCC-3' and reverse primer of 5'-CAGTGAAGAGGTGT AGCCGC-3') was designed by Gene Runner (version 5.1) software and synthesized by Macrogen (South Korea). PCR reaction mixture was contained 1X PCR buffer, 2 mM MgCl2, 2.5 unit of Taq DNA Polymerase , 0.5 mM dNTP, 5 pM of each primer, and 30 ng of pattern DNA up to the volume of 25 μl of sterile distilled water. Ampliﬁcation was performed as follows: initial denaturation at 95oC for 5 min, then 30 cycles of denaturation at 94oC for 30 s, annealing at 60oC for 45 s, extension at 72oC for 60 s and final extension at 72oC for 10 min.Genomic DNA was extracted from 5 mL of EDTA anticoagulated whole blood using AccuPrep® Genomic DNA Extraction Kit  based on manufacturer's manual. To study MspI  for 16 hr at 37ºC. Then MspI restriction digestion products were loaded on 2% agarose gel. The genotypes were characterized as TT when the PCR products remained uncut (343 bp), as CC when the restriction site was present, and as TC when the digestion produced a pattern of three DNA fragments sized 343 bp, 136 bp, and 207 bp.The ampliﬁcation products (343 bp) were digested with Ethical considerationThe Islamic Azad University, Yazd Branch Ethics Committee approved this study . Written informed consent was obtained from all participants. Statistical analysis2 or Fisher's exact test was used to compare genotype distributions between patients and controls. The statistical modeling using logistic regression was used to calculate the relative risk  of genotypes for case-control study. Odds ratios were expressed together with the 95% conﬁdence interval . The mean body mass index (BMI) was 24.30±3.69 in the case group and 26.14±4.82 in controls. Statistical analysis showed that BMI of the cases is significantly less than control group (p=0.003) . Stage I2 test revealed that there is no significant difference between the two groups (p=0.961). Using the TT homozygote as a reference group, the OR of TC heterozygotes  and CC homozygotes was . Allele frequencies of MspI polymorphism in the case and control groups are shown in 2 test indicates no significant difference between two groups (p=0.804). In all stages of endometriosis, CC genotype has the lowest frequency of 7.2%, 5.4%, 0.0%, and 4.5% for stages I to IV, respectively (MspI genotypes by Fisher's exact test indicated that there is no significant difference between the genotypes and different stages of endometriosis (p=0.491). In addition, the patients with advanced stages were compared with the primary stages and control group; there was no significant difference between two groups .The most frequent genotype in two groups was TT. The frequencies of this genotype in patients and control group were 73.1% and 74.1%, respectively. Data analysis by χectively . StatistCYP1A1 gene in metabolism of estrogen and PAH has been well-defined  . Previous reports on association of (p=0.11) , China ((p=0.11) , India ((p=0.11) , 23, Aus(p=0.11) , Taiwan . Previou(p=0.11)  and Braz(p=0.11) . In our (p=0.11) , 21-24. (p=0.11) , 25, 26.CYP1A1 MspI polymorphism was not associated with endometriosis risk in our population. More studies with larger sample size and especially, in other populations with different ethnicity can provide a better understanding of the relationship between population-specific environmental variables and genetic backgrounds.In conclusion, this study showed that


To find the perceived stress level and monetary burden in the thalassemia patients and their caregivers related to thalassemia treatment sessions.A descriptive cross sectional study was conducted at Rawalpindi Thalassemia Centre and Military Hospital Rawalpindi of six months’ duration from November 2016 to April 2017. A total of 87 sample size was calculated by using WHO sample size calculator. Participants were inducted through purposive sampling technique from thalassemia centers. A validated standardized Cohen’s Perceived Stress score was used. Data were entered and analyzed in SPSS 22.Mean age of the participants was 30.42 ± 14.53 years. There were 30 (34.2%) males and 57 (65.8%) females. There were 39 (51.3%) patients and 48 (48.7%) caregivers. The mean income per month of the participants was 48706.9 ± 39492.68 PKR. The mean total expenditure per treatment session was 48706 ± 4037.12 PKR. Among the patients, there were 15 (38.5%) who were having average stress, while 4 (10.3%) were having moderate stress while 45  were having mild stress, 13 (27.1%) were having moderate stress while 25 (52.1%) were having severe stress. The stress among the two groups was not statistically significant (p=0.066).More than half of the patients and caregivers were having a high perceived stress levels and there was a significant association between the two groups. The monetary burden was a lot to bear by the patients and the cost of treatment session most expensive. Thalassemia is a haemoglobinopathy and specifically an autosomal recessive disorder. The word thalassemia is derived from the Greek word thalassa which means “sea”, and Latin word - emia (from Greek haema) meaning “blood”.2In a normal person there is coordinated synthesis of the α- and β-globin chains, that is for each α-globin chain, a β-globin chain will be present consequently normal hemoglobin α2β2 (HbA) will be formed. However in thalassemia there is defective synthesis of either the α- or the β-globin chain.4Thalassemia though a preventable disease has an alarmingly high global prevalence. Prevalence of α-thalassemia in various region is: America 0-5%, Europe 1-2%, Eastern Mediterranean 0-2% and South East Asia 1-30% while prevalence of β-thalassemia is: America 0-3%, Eastern Mediterranean 2-18% and South East Asia 0-11%.7The disease itself has high morbidity and mortality which is augmented by the high frequency of treatment. Prevalence of complications is: Heart Failure 6-8%, Arrhythmias 5-7%, Hypogonadism 54.7%, Hypothyroidism 10.8%, Diabetes 6.4%, HIV infection 1.7% and thrombosis 1.1%.9Stress can be defined as any uncomfortable or agonizing emotional experience associated with changes in behavior as well as biochemical and physiological changes or in simple words we can say it is a feeling of being worried or overwhelmed. It can affect anyone regardless of age or gender of person. Stress can result in both physical as well as psychological health issues.10Stress in thalassemia patients and their parents can be attributed to a number of reasons like frequent treatment procedures and hospital visits, decreased life expectancy, expected complications from disease or treatment procedures and the monetary burden on parents/guardian.Considering the fact that thalassemia is not a curable disease thus all that can be done is to improve the quality of life of the patient and decrease the morbidity . Stress both in patients and their caregivers, which in our setup are mainly parents, is the biggest hindrance in improving the quality of life along with monetary issues. The aim of this study was to explore in which category of stress the thalassemia patients and their caregivers are? This will in the long run help policy makers, clinician and community to focus on stress and the monetary burden among thalassemia affected families and thus appropriate interventions can be planned and executed in this neglected but grave aspect of disease.th Year MBBS students after taking informed consent collected the data from the two centres. Data were collected from patients during the blood transfusion period. The data collection tool was a closed ended questionnaire. The questionnaire was translated into Urdu and verified by two subject specialists. It had two parts, first part was related to participant’s demographic details and monetory burden in terms of Pakistani rupees.This was a descriptive cross sectional study conducted in Rawalpindi Thalassemia Centre and Military Hospital Rawalpindi. The duration of study was six months i.e. November 2016 till April 2017. A sample size of 87 was calculated by using Openepi info with hypothesized % frequency of thalassemia in population (p=1000) as 6% and with confidence limits as 5%. Non-probability purposive sampling was employed for the study. Thalassemia patients and their caretakers who usually assisted them for their treatment sessions and were responsible for their management and daily activities were included while those patients and caregivers who didn’t give consent, patients having any co-morbid conditions were excluded. This research was approved by ethical review committee of Army Medical College and informed voluntary consent was taken before starting interview and no risk or harm was anticipated to the best of our knowledge. Trained 4The participant were divided into two group i.e patients and their caregivers. The second part was related to stress evaluation by validated standardized Cohen Perceived Stress question aire.The total number of participants were 87 people, out of which 39 were patients and 48 were caregivers. The mean age of the patient was 17.09 ± 4.4 years while of the caregivers was 41.25 ± 10.17 years with statistically significant difference (p<0.001). Out of 39 patients, 16 (41%) were males and 23 (59%) were females whereas out of 48 caregivers, 14 (29.2%) were males and 34 (70.8%) were females.Regarding education status, among patients, there were 10 (11.5%) illiterate, 24 (27.6%) having primary education, 37 (42.5%) having secondary education and 16 (18.4%) having higher level education. Among the caregivers, 4 (10.3%) were illiterate, 14 (35.9%) having primary education, 19 (48.7%) having secondary education and 2 (5.1%) having higher level of education. There was a statistically significant association among the two group’s educational status (p=0.026).The mean income per month of the participants was 48706.9 ± 39492.68 PKR. The mean total expenditure per treatment session was 48706 ± 4037.12 PKR. Out of the total expenditure, the treatment, transport, food, accommodation and others miscellaneous cost were 2257.99 ± 3905.2 PKR, 764.94 ± 818.68 PKR, 325.29 ± 3905.2 PKR, 28.74 ± 139.69 PKR and 256.32 ± 1012.56 PKR respectively. Details are shown in Among the patients, there were 15 (38.5%) who were having mild stress, while four (10.3%) were having moderate stress 45 (51.7%) were having severe stress. Among the caregivers, 10 (20.8%) were having mild stress, 13 (27.1%) were having moderate stress while 25 (52.1%) were having severe stress. The stress among the two groups was not statistically significant (p=0.066). As shown in Thalassemia is a chronic disorder that causes illness in children and impacts their families, complications of invasive treatment procedures and monetary burden causes physical, mental, emotional and psychological suffering.Monetary burden also adds to the stress experienced by caregivers and this monetary burden can be attributed to treatment, transport, stay, medicines and food expenditures.24In our study perceived stress score of patients came out to be severe in 51.28%, moderate level of stress among 10.25% and mild stress in 38.46%.According to Greek Transfusion center, severe depressive symptoms were found in 17 patients (15%), anxiety symptoms in 11(9.6%) and stress symptoms in 35(30.2%).6Parents of thalassemia patients have to face higher levels of distress which affects the lifestyle and coping style of caregivers. Psychiatric morbidities aren’t limited to the patients, in fact they extend to the caregivers as well.15This study was conducted only in two centers, Thalassemia Center Rawalpindi and Military Hospital RWP which limits its generalization. Purposive sampling technique was used for this study and so the study results does not depict a true representation of the whole population.About half of the thalassemia patients and their caregivers who participated in the study had severe perceived stress and stress was more marked in caregivers as compared to patients. The monetary burden related to thalassemia treatment session was too much to bear by the patients and their caregivers, treatment cost being most expensive.On the basis of results of this study, we recommend that a holistic approach should be taken in treatment of Thalassemia with special attention to the caregivers including their counselling. The health care practitioners and physicians should be mindful of the psychological and emotional implications of this disease on the parents and their caregivers. Parents and caregivers should be properly counseled and educated about the disease and their apprehensions should be addressed. If the need is felt, psychiatric referral should be considered for severely stressed patients and their caregivers.As far as the financial aspect of the disease is concerned it is also a contributory factor in the stress experienced by the caregivers so maximum relief should be provided in this regard, in form of government subsidies and financial assistance by NGOs. Hospitals can play their part by offering subsidies on blood bags, medications and other possible compensation packages.In addition to all this, efforts should also be directed towards prevention of thalassemia by measures like premarital screening of thalassemia minor trait and appropriate counseling in case of detection to decrease the incidence of the disease and consequential psychological and economic burden.HA: Designed and did statistical analysis & final editing of manuscript.NSK & TNA: Conceived the study, designed methodology and manuscript writing.IH, AB & NM: data collection, discussion writing and review of manuscript.


Adipocyte fatty acid–binding protein (FABP4) played critical roles in metabolic syndrome, inflammatory responses and cardiovascular diseases. It aimed to investigate the associations of serum FABP4 levels with early stroke recurrence. This study included the 206 acute ischemic stroke patients hospitalized in our institution. Stroke recurrence events were assessed at the 3-month follow-up. The median of FABP level was 22.6  ng/mL in patients with stroke recurrence (N=36), which was higher than in patients without stroke recurrence [16.9  ng/mL] (P<0.001). As a continuous variable, the unadjusted and adjusted risk of stroke recurrence would be increased by 12%  and 8%  for every 1 ng/ml increment of FABP4. The Area under the curve (AUC) of serum FABP4 and NIH Stroke Scale (NIHSS) score for predicting stroke recurrence was 0.73 (95% CI: 0.64–0.82) and 0.72 (95% CI: 0.64–0.81), presenting no discriminating capacity (P=0.45). In the combining model, the AUC of NIHSS score was further improved to 0.77 by FABP4 , which was significant (P=0.01). The risk of stroke recurrence can be predicted by elevated FABP4 levels in serum of nondiabetic patients with first-ever ischemic stroke. Stroke has been the cause of approximately 5% of all disability-adjusted life-years, as well as 10% of all mortality globally . FurtherAdipocyte fatty acid–binding protein FABP4/aP2) has been reported to function in metabolite and inflammation /aP2 has , insulinInterestingly, one study reported that serum FABP4 levels in elderly population were related to an elevated risk of developing central arterial stiffness . A commu324 patients with ischemic stroke were included. 255 patients were recorded after excluding 45 with diabetes mellitus, 8 with tumors, 6 inflammatory disease and 10 with abnormal renal and liver function. There were 9 lost to follow-up and 5 withdrew. Finally, 241 patients completed follow-up and 206 patients were included (35 patients died during follow-up). The FABP levels of those patients were collected and analyzed. The median value of FABP levels was calculated as 17.7  ng/ml and the median value of age was 59  years. The median NIHSS score was 7  points during their hospitalization. The baseline information of all patients was summarized .A positive relationship was observed between serum FABP4 levels and stroke severity, since the stroke severity was evaluated with NIHSS score , Stroke recurrence was observed in 36 patients . Median of serum FABP4 levels in patients with stroke recurrence was 22.6  ng/mL, which was higher than those of without stroke recurrence  . The difThe distribution of stroke recurrence across the FABP4 quartiles was ranged from 5.8% (Q1) to 34.0% (Q4) . For strThe discriminating capacity of FABP4 for predicting stroke recurrence was evaluated with AUC . The result was compared with Hs-CRP , HCY  and NIHSS score  . InteresThe FABP4-improved discriminating capacity was further confirmed with the internal 5-fold cross validation. The AUC (mean ± SD) was 0.73± 0.046 and 0.77± 0.040 for the NIHSS and the combining model, respectively, amount to a difference of 0.04± 0.006). The 5-fold cross-validated mean squared prediction error was 0.209 ± 0.014 and 0.196 ± 0.014 for the NIHSS and the combining model, respectively, amount to an average decrease of 0.013 ± 0.004). Further, higher discriminating capacity was observed in a model combining known risk factors and FABP4, compared to the model only involving known risk factors without FABP4 (P=0.009).In the analysis, those patients with endpoint of all-cause death (N=35) were taken into account. A significantly higher discriminating capacity was still observed for FABP4 to predict stroke recurrence , compared to Hs-CRP , HCY  and NIHSS score .2) and non-obese. In the multivariate analysis, the date showed that for each 1 ng/ml increase of FABP4, the association between FABP4 and stroke recurrence was stronger among patients who defined as obese  versus non-obese .In addition, we also conducted analyses separately among patients who defined as obese  and single center cannot produce effective results. In addition, there were only 36 recurrent strokes, leading to a relatively limited power. Thus, the adjustment in logistic regression may be too extensive with overfitting. Second, circulating FABP4 levels were determined with a single measurement at baseline. Without serial testing of FABP4 levels, we yielded no data regarding the change of FABP4 levels in those nondiabetic stroke patients. Interestingly, one study reported significantly elevated FABP4 levels during the early hours after the occurrence of acute myocardial infarction . In addiIn summary, this cohort study was performed on nondiabetic patients with ischemic stroke. The results indicated that increased FABP4 level in serum was related to higher risk of early stroke recurrence in the future, independent of baseline variables. Because it makes critical effects at multiple stages of stroke development, FABP4 will be worthy of further research as a possible therapeutic target.During December 2016 and May 2018, this study included the consecutive patients hospitalized in Beijing Tian Tan Hospital, Capital Medical University (China) due to the acute ischemic stroke. Acute ischemic stroke was determined per the recommendations of World Health Organization recommendations  . The dia2), vascular risk factors . Median follow-up was 3 months. Stroke recurrence was the primary endpoint, defined as suddenly deteriorated neurological function evaluated as a decreased NIHSS score of 4 or more, or a new focal neurological deficit of vascular origin lasted for >24 hours . All-cauFasting blood of patients were drawn at 8:00 on the first hospitalization day for testing levels of FABP4. The serum FABP4 was detected with a commercial ELISA kit . For this assay, the determination range was 1.5-100 ng/ml. The coefficient of variation intra- and inter-assay was less than 10.0% in the range of 100-10 ng/ml. In addition, other serum biomarkers were also detected, such as C-reactive protein (Hs-CRP) and homocysteine (HCY).The results were expressed as percentages for categorical variables and as medians (interquartile ranges [IQRs]) for continuous variables. The comparison was conducted with Mann-Whitney U test for continuous variables and with chi-square test for categorical variables. Bivariate correlation was assessed by the Spearman's rank correlation.st quartile as reference. The multivariate analysis model was applied for further estimating the adjusted OR (95%CI). Further, the relationship between high FABP4 (defined as >3rd quartile=22.8ng/ml) and stroke recurrence was also presented.The association between FABP4 and stroke recurrence was analyzed with logistic regression models. The odds ratios  were analyzed with both unadjusted and adjusted model. In addition, the association between stroke recurrence and FABP4 quartiles was explored, with the 1Second, the risk of stroke recurrence obtained with various models were compared. The receiver operating characteristic curve (ROC) was plotted and the area under the curve (AUC) was calculated for obtaining criteria and cut-point. To test if the efficiency could be improved with FABP4 level, two nested logistic regression models were applied. In addition, optimistic bias of in-sample prediction error estimates was carefully adjusted with a fivefold cross-validation scheme .http://cran.r-project.org/). P<0.05 was considered to be significant.The statistics were analyzed with SPSS, version 22.0. The ROC was plotted with ROCR package, version 1.0-2 (CRAN repository, This study has been reviewed and approved by the ethics committee of the Beijing Tian Tan Hospital, Capital Medical University. The study protocol was introduced and interpreted to all the patients and informed consents of all the involved patients were obtained.Written informed consents were obtained from all patients; and, this study conformed to the principles of the Declaration of Helsinki was approved by the investigational review board of the Beijing Tian Tan Hospital, Capital Medical University.Please contact author for data requests.


Cinchona bark still play an important role in medicine, for example as anti-malarial and antiarrhythmic drugs. For the first time Supercritical Fluid Chromatography has been utilized for their separation. Six respective derivatives  could be resolved in less than 7 min, and three of them quantified in crude plant extracts. The optimum stationary phase showed to be an Acquity UPC2 Torus DEA 1.7 µm column, the mobile phase comprised of CO2, acetonitrile, methanol and diethylamine. Method validation confirmed that the procedure is selective, accurate (recovery rates from 97.2% to 103.7%), precise  and linear (R2 ≥ 0.999); at 275 nm the observed detection limits were always below 2.5 µg/ml. In all of the samples analyzed cinchonine dominated (1.87%–2.30%), followed by quinine and cinchonidine. Their total content ranged from 4.75% to 5.20%. These values are in good agreement with published data, so that due to unmatched speed and environmental friendly character SFC is definitely an excellent alternative for the analysis of these important natural products.Chinoline alkaloids found in  It became the primary remedy against this disease worldwide, and only after World War 2 synthetic antimalarials like chloroquine replaced the natural product [Cinchona bark is rather complex with more than 30 known representatives [Cinchonae cortex, which originates from several related species of the genus  product . However product , serves  product , or acts product . The alkntatives . They maCinchona bark but also the narrow therapeutic window of quinine many analytical studies focused on the determination of alkaloids in the crude drug. The compendial method in the 9th edition of the Ph.Eu. is based on a photometric determination of quinine (348 nm) and cinchonine-type (316 nm) alkaloids. Research papers mainly emphasized on the separation of the dominant representatives utilizing TLC [Cinchona alkaloids in 15 min, yet an application to plant material is missing [Cinchona bark by SFC.Not only due to the medicinal and commercial importance of zing TLC , isotachuinine 34 nm and czing TLC , vibratizing TLC , NMR [12e-type 31 nm alkalzing TLC –16. For  missing . Whether missing , triglyc missing  or carot missing . Recent  missing ,22 and a missing –25. Ther22.1Cinchona alkaloids  and Sigma Aldrich . Plant samples (CC-2017-1 to CC-2017-4) were bought 2017 in different pharmacies in Innsbruck, Austria; voucher specimens are deposited at the Institute of Pharmacy, Pharmacognosy, University of Innsbruck. Compressed carbon dioxide for SFC analysis had a purity of ≥99.995% (4.5 grade) and came from Messer . All solvents and reagents  utilized in this study were of analytical grade and purchased from Merck . An Arium 611 water purification system from Sartorius  produced the required HPLC grade water.Six 2.2g, and the clear supernatant combined in a 50 ml volumetric flask. Then the latter was filled to volume with the extraction solvent. Sample solutions were membrane filtered right before analysis  and injected in triplicate. If stored at 4 °C sample and standard solutions are stable for at least 2 weeks.The plant material (Cinchonae cortex Ph.Eu.) was finely pulverized in a mill and 150 mg were extracted following a published protocol . Extract2.32-SFC instrument from Waters , equipped with convergence manager, column oven, sample manager, binary solvent manager and PDA detector was used; the operating software was Empower 3. Optimum separation of the six standards was achieved on an Acquity UPC2 Torus DEA column  from Waters, protected by a guard filter . The mobile phase comprised CO2 (A) and 0.8% diethylamine in a mixture of 10% acetonitrile and 90% methanol (B). Isocratic separation was achieved by maintaining a concentration of 97.7A/2.3B over 10 min. The injected sample volume was 1 µl, while flow rate, column temperature and ABPR pressure were set to 1.8 ml/min, 15 °C and 150 bar (2175 psi). The compounds of interest were detected at 275 nm. The sample manager was maintained at 10 °C, and a mixture of methanol/2-propanol (1:1) and methanol served as a weak and strong wash, respectively.For all experiments an Acquity UPC2.4To assure that the developed SFC method conforms to regulatory standards it was validated according to ICH guidelines . For the3Cinchona bark, a drug which is analytically challenging as it contains diastereomeric chinoline alkaloids as active constituents.Since its beginnings in the 1960s SFC has evolved into a widely utilized and efficient separation technique. A better understanding of the underlying theory, together with significantly improved instruments and stationary phases have led to many successful separations and a broad field of applications. However, relevant medicinal plants, whose ingredients seem to be not suitable for SFC because of their polarity, have never been investigated till date. One of them is 3.1Cinchona alkaloids, namely dihydroquinidine (1), dihydroquinine (2), quinidine (3), quinine (4), cinchonine (5) and cinchonidine (6), within less than 7 min is shown in The optimum SFC separation of six major 2 is similar to hexane [2) and 2.3% B  provided the best resolution. It is note-worthy to say that already a slight change  to 2.4 (2) µg/ml, LOQ values varied from 1.9–7.3 µg/ml. They naturally cannot compete with those achievable by fluorescence detection  and not higher than 103.7% , indicating validity of this parameter too.Assay development was followed by method validation; data presented in  2 fmol, ); howeve 2 fmol, . Precisi3.3Cinchona bark, all of them with Ph.Eu. quality, were available for quantification. Concerning the optimum extraction protocol a procedure described by Gatti et al. was adopted [Four samples of dried and milled  adopted . It utilrel ≤ 1.55, n = 3). The total alkaloid content varied from 4.75% (sample CC-2017-3) to 5.20% (sample CC-2017-2).A typical SFC chromatogram of a sample solution is shown in 4Cinchona bark is a challenging task, because the target analytes are structurally very similar and the investigated matrix is complex like most biological samples. Due to the persisting practical relevance of the drug many attempts have been made to determine these compounds, mostly by using conventional RP-HPLC in combination with fluorescence detection. This assured an excellent sensitivity; however, the required analysis time was in the range from 15 [Cinchona bark extracts. The quantitative results were well comparable to published data, which for example report the following values for a drug with Ph.EU. quality: 1.80% quinine, 1.65% cinchonine, and 1.25% cinchonidine [This study is another proof for the excellent separation efficiency and versatility of SFC, especially in the field of natural products. The determination of alkaloids in  from 15  to 50 mi from 15 , when onhonidine . This suhonidine , kavalachonidine  or furochonidine  are goodhttps://doi.org/10.1016/j.chroma.2018.04.038.Supplementary data associated with this article can be found, in the online version, at Supplementary Information


According to studies by the National Kidney Foundation and Food and Drug Administration, 30% and 40% declines in estimated glomerular filtration rate (eGFR) could be used as surrogate endpoints of end-stage renal disease (ESRD). However, the benefits of using these endpoints in diabetic patients remain unclear.This cohort study comprised Japanese patients with type 2 diabetes; those with repeated serum creatinine measurements during a baseline period of 2 years (n = 1868) or 3 years (n = 2001) were enrolled. Subsequent risks of ESRD following eGFR declines were assessed.In the 2-year baseline analysis, the cumulative prevalence of −20%, −30%, −40%, and −53% changes in eGFR were 23.9%, 11.1%, 6.8%, and 3.7%, respectively. There were 133 cases (7.1%) of subsequent ESRD during a median follow-up period of 6.5 years. In the 3-year baseline analysis, the corresponding proportions were 28.1%, 14.0%, 7.7%, and 3.9%, respectively, with 110 participants (5.5%) reaching ESRD during a median follow-up period of 5.5 years. The adjusted hazard ratios of subsequent ESRD following −53%, −40%, −30%, and −20% changes in eGFR during the 2-year baseline period were 22.9 (11.1–47.3), 12.8 (6.9–23.7), 8.2 (4.3–15.5), and 3.9 (2.2–7.0), respectively when compared with the no changes in eGFR. In the 3-year baseline analysis, the corresponding risks were 29.7 (10.8–81.9), 18.4 (7.6–44.7), 12.8 (5.2–32.2), and 5.4 (2.3–12.8), respectively. In the subgroup analysis, similar trends were observed in patients with macroalbuminuria at baseline.Declines in eGFR were strongly associated with subsequent risk of ESRD in Japanese type 2 diabetic patients. In addition to 30% and 40% declines, a 20% decline in eGFR over 2 years could be considered as a candidate surrogate endpoint of ESRD in diabetic kidney disease. Diabetes mellitus is the leading cause of chronic kidney disease (CKD) worldwide . In factSeveral clinical trials using established endpoints such as kidney failure and doubling of serum creatinine (sCr) level, which corresponds approximately to a 57% decline in eGFR using the 2009 CKD-EPI (CKD Epidemiology Collaboration) creatinine equation, have been conducted . HoweverRecently, the National Kidney Foundation (NKF) and Food and Drug Administration (FDA) committee has published a series of studies to determine whether lesser eGFR declines can be used as a substitute for the doubling of sCr levels among composite kidney endpoints , 12–14. In Japanese patients with reduced eGFR, 30% and 40% declines over a period of 1 to 2 years were reported to be strongly associated with the risk of ESRD . In the This cohort study comprised 4814 Japanese patients with type 2 diabetes, who were treated at 10 centers between 1985 and 2010. We diagnosed type 2 diabetes mellitus according to the Japan Diabetes Society (JDS) criteria [At the beginning of the baseline period, baseline assessment of patients’ characteristics including laboratory measurements , physical findings, sociodemographic information, and medical history were performed at each center.Blood pressure was measured in the sitting position. sCr was measured by an enzymatic method and eGFR was estimated using the equation proposed by the Japanese Society of Nephrology . Urine aBased on the classification of CKD , the albhttp://www.who.int/classification/icd/en/). Participants were followed until the onset of the first event or the last day of the observation period after each baseline period.The primary outcome of this study was ESRD, which was defined as the initiation of dialysis or kidney transplantation at each clinical center through 2010. These conditions corresponded to the International Classification of Diseases, 11th version . Data are expressed as mean ± standard deviation (SD).We estimated the adjusted hazard ratios (HRs) of subsequent ESRD from models with a spline function of percent changes in eGFR and covariates including age, gender, baseline eGFR, baseline UACR, baseline systolic BP, and a history of CVD. Based on previous reports , 12, 17,The positive predictive value (PPV) was then calculated for each surrogate endpoint. P values were two-sided and those <0.05 were considered statistically significant. All analyses were performed using Stata/IC version 15.0 software .http://www.lifescience.mext.go.jp/files/pdf/n796_01.pdf). The study was approved by the Kanazawa University ethical committee .This study was conducted according to the ethical guidelines for epidemiological research designed by the Japanese Ministry of Education, Culture, Sports, Science and Technology and Ministry of Health, Labor, and Welfare  was 20  mg/gCr. In the 3-year baseline analysis, the mean age was 60 years, 57.9% were men, mean eGFR was 77 ± 24 mL/min/1.73 m2, and median UACR was 19  mg/gCr.th–95th percentile; −48% to +14%) and −13% (−48% to +12%), respectively. The distribution was negatively skewed , 11.1% (207 patients), 6.8% (127 patients), and 3.7% (70 patients), respectively , 14.0% (281 patients), 7.7% (155 patients), and 3.9% (79 patients), respectively  during the median follow-up of 6.5 years and the overall incidence rate of death was 8.1 per 1000 person-years. Among the 129 cases with all-cause mortality, 15.5% (20 cases) and 3.1% (4 cases) reached ≤−30% and ≤−53% changes in eGFR, respectively. In the 3-year baseline analysis, there were 128 (6.4%) death during the median follow-up of 5.5 years and the overall incidence rate of death was 7.3 per 1000 person-years. The risks for all-cause mortality are shown according to the achieved percent changes in eGFR during the 2-year  or 3-yeaIn this study, we examined the possibility of a decrease in eGFR as a surrogate endpoint for ESRD in Japanese type 2 diabetes patients over a baseline period of 2 or 3 years. In 2014, the NKF-FDA committee proposed 30% and 40% declines in eGFR as novel surrogate endpoints for CKD progression in clinical trials, based on the results of some meta-analyses and simulation studies , 12–14. The findings of the current study are in accordance with those reported in several previous studies; we have previously published the clinical impact of reduced eGFR on renal events in type 2 diabetic cohort studies –25. The The present study has an advantage in providing analysis stratified by albuminuria at baseline, by conducting a large-scale research focusing on diabetic patients. We observed that eGFR declines were considerably related to ESRD risk in type 2 diabetes patients with macroalbuminuria. This observation is consistent with other studies, which reported that the rapid decline in eGFR could predict the risk of ESRD in type 1 diabetes patients with macroalbuminuria –29. On tWe estimate that the possibility of surrogate endpoints obtained in the current study can be applied to diabetic patients all over the world, especially to Asian patients who show a high prevalence of albuminuria , 33. TheDeclines in eGFR occurred more commonly and were associated with the risk of mortality, as previously reported . In the There are several limitations in this study. Firstly, the number of the patients is relatively small, especially in the subgroup analysis. Therefore, the usefulness of surrogate endpoints in patients with normoalbuminuria and microalbuminuria may be unclear due to insufficient detection power. Secondly, although we adjusted for multiple confounding factors, we cannot exclude residual confounding such as body mass index, smoking status, and medications. Especially, in the current study, there is lack of data regarding the medications for blood pressure and glucose control to analyze the treatment effects. In addition, variability in sCr measurements among centers must be considered; moreover, we did not measure sCr regularly. These issues can lead to less accurate calculations of the percent changes in eGFR and reduce the statistical power of the analyses. Thirdly, although this study excluded people who reached ESRD within the 2- or 3-year baseline period from the analysis, we did not distinguish participants who chronically achieved ESRD from those who had acute kidney injury in the follow-up periods, which might result in somewhat imprecise inference about the relationships between chronic changes in eGFR and ESRD. However, the ability to demonstrate the usefulness of surrogate endpoints in a situation close to the actual clinical site indicates its robustness.In conclusion, our study revealed that eGFR declines were strongly associated with subsequent risk of ESRD in Japanese type 2 diabetic patients. In addition to 30% and 40% declines in eGFR, a 20% decline in eGFR over 2 years could be considered as a candidate surrogate endpoint of ESRD in DKD. These results were confirmed mainly in patients with macroalbuminuria. In future, simulation studies, as described in the accompanying articles ,14, may S1 FigAbbreviations: UACR, urine albumin-to-creatinine ratio; BP, blood pressure; ESRD, end-stage renal disease; sCr, serum creatinine.(TIFF)Click here for additional data file.S1 TableBaseline characteristics according to percent changes in eGFR during the (a) 2-year or (b) 3-year baseline period.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.