Low- and high-resolution scattering length density models for the joint analysis of small-angle neutron and X-ray scattering experiments on asymmetric lipid vesicles are presented. i.e. above the melting transition temperature of the two lipids.Low- and high-resolution models describing the internal transbilayer structure of asymmetric lipid vesicles have been developed. These models can be used for the joint analysis of small-angle neutron and X-ray scattering data. The models describe the underlying scattering length density/electron density profiles either in terms of slabs or through the so-called scattering density profile, previously applied to symmetric lipid vesicles. Both models yield structural details of asymmetric membranes, such as the individual area per lipid, and the hydrocarbon thickness of the inner and outer bilayer leaflets. The scattering density profile model, however, comes at a cost of increased computational effort but results in greater structural resolution, showing a slightly lower packing of lipids in the outer bilayer leaflet of ∼120 nm diameter palmitoyloleoyl phosphatidylcholine (POPC) vesicles, compared to the inner leaflet. Analysis of asymmetric dipalmitoyl phosphatidylcholine/POPC vesicles did not reveal evidence of transbilayer coupling between the inner and outer leaflets at 323 K,  For example, mammalian plasma membranes contain mainly phosphatidylcholine (PC) and sphingomyelin in their outer (exoplasmic) leaflets, while phosphatidylserine and phosphatidylethanolamine are the major lipid groups of their inner (cytosolic) leaflets -mediated lipid exchange for producing solvent-free free-floating asymmetric vesicles of different sizes (Cheng i.e. noncentrosymmetric) lipid vesicles. Later, Kučerka and co-workers described an SDP-based model for asymmetric bilayers  and dipalmitoyl phosphatidylcholine (DPPC), as well as their deuterated variants. Despite the significant difference in spatial resolution, the two models yield comparable values for the area per lipid 3.3.1.Fig. S6 of the supporting information for details of chemical structures). DAll lipids, including their isotopes  were purchased from Avanti Polar Lipids  and used without further purification . The resulting multilamellar vesicles (MLVs) were incubated at 313 K for 1 h with intermittent vortex mixing, followed by five freeze/thaw cycles using liquid nitrogen. LUVs were prepared using a hand-held mini-extruder  with a 100 nm pore-diameter polycarbonate filter. The MLV suspension was passed through the filter a total of 31 times at room temperature. LUV formation was facilitated by doping the lipids with 5 mol% POPG or POPG-d31 (matching the isotopic composition of the inner leaflet POPC or POPC-d31 lipids). Such low amounts of the charged lipid were previously shown to not affect the membrane structure of phosphatidylcholines  composed of the outer leaflet lipids were prepared by hydrating the dry lipid films in water containing 20%(w/w) sucrose using vortex mixing in combination with three freeze/thaw cycles. Donor MLVs were then diluted 20-fold with water and centrifuged for 30 min at 20 000 g in order to remove extravesicular sucrose. The resulting pellet was re-suspended in a 35 mM mβCD solution at a lipid:mβCD ratio of 1:8 and incubated for 2 h at room temperature, while being gently stirred.Donor multilamellar vesicles  was then concentrated to <0.5 ml with a centrifugal ultrafiltration device . The initial concentration step was followed by three cycles of successive dilution to 11 ml and concentration to <0.5 ml, effectively removing residual CD and sucrose, and allowing for the exchange of Het al.  instrument of the Spallation Neutron Source, located at Oak Ridge National Laboratory (ORNL), and KWS-1 at the FRM II reactor   scattering curves using Mantid .Neutron scattering experiments were performed at the BL-6 extended-3.3.et al., 201512 s−1 focused to a spot of 120 × 200 µm . The combination of 20 keV  was used for data collection. SAXS data from DPPC/POPC aLUVS were obtained at the ESRF BM29 BioSAXS beamline  or ten (BM29) frames were recorded with an exposure time of 0.095 s (P12) or 0.5 s (BM29). The water background was measured before and after each sample. To determine the occurrence of possible radiation damage, data collected in subsequent frames were compared by a standard F-test  in Hamburg, Germany . Equation (1)et al., 2006−1, and the scattered intensity can be approximated by It was shown previously , which essentially couples the thicknesses of the individual layers to the projected area per lipid supporting information (§2). The number of headgroup-bound water molecules, et al., 2006et al., 2008et al., 1997et al. (2011Ds in equation (4)supporting information). Alternatively, Following the approach used by Kučerka and co-workers . In order to account for this, we approximated the A complication we encountered was that the different contrast aLUV preparations showed a small but non-negligible variation of outer leaflet exchange Using similar arguments as in §4.1.1et al., 2011et al.  of quasi-molecular lipid fragments. The VPPs are scaled by either the fragment’s total coherent neutron scattering length (in the case of SANS) or the number of electrons (in the case of SAXS) to obtain the SLD profile  probability function For the real part of et al., 2011i.e.et al., 2011et al.  parameters, number of iterations and termination tolerances.5.5.1.i.e. POPC, POPC-d13, POPC-d31 and POPC-d44). SANS data were taken from the work of Heberle et al. . SANS data previously reported by Heberle et al. , we conclude that these scenarios cannot be distinguished owing to the inherent resolution limitations of the slab model.Fig. 5s Table 2, as wellet al., 2016In the case of the aSDP model, unconstrained fits led to 5.3.i.e. DPPC-d62Fig. S2. A previous analysis of outside gel/inside fluid DPPC/POPC aLUVs at room temperature showed a partial fluidization of the outer leaflet, observed as a significantly larger et al., 2016i.e. above the melting temperature of both lipids.Fig. 6Table S6). Data were therefore analyzed according to equations (8)i.e. in terms of the individual structures of DPPC and POPC lipids.Analysis of the aLUV composition yielded significant differences in samples with different contrasts (2) and POPC (2) were also consistent with those from a previous analysis of single-component bilayers at 323 K . Additionally, we applied a DE algorithm to prevent the optimization routine from becoming trapped in local minima. In the case of the slab model, which has fewer free parameters, the trust region reflective algorithm was sufficient. On the basis of the fit quality, as judged by Application of the aSDP model requires a large number of adjustable parameters, some of which were constrained in order to avoid nonphysical results  that are needed to understand the complex structure of asymmetric membranes on the sub-nanometre level.Both models were tested on isotopically labeled variants of POPC and DPPC/POPC aLUVs. Interestingly, our analysis of POPC aLUVs suggested 7.supporting information, see Hartler et al. (2011et al. (2014et al. (1985For further literature related to the  al. 2011, Knittel al. 2014 and Perl al. 1985.10.1107/S1600576717000656/vg5060sup1.pdfSupporting information file. DOI:


Mesenchymal stem cell transplantation (MSCT) became available with liver failure (LF), while the advantages of MSCs remain controversial. We aimed to assess clinical advantages of MSCT in patients with LF.  Clinical researches reporting MSCT in LF patients were searched and included. n = 476) related with LF patients were enrolled. After MSCT, alanine aminotransferase (ALT) baseline decreased largely at half a month (P < 0.05); total bilirubin (TBIL) baseline declined to a certain stable level of 78.57 μmol/L at 2 and 3 months (P < 0.05). Notably, the decreased value  of Model for End-Stage Liver Disease score (MELD) of acute-on-chronic liver failure (ACLF) group was higher than that of chronic liver failure (CLF) group . Moreover, MELD baseline of ≥20 group was a higher D value of MELD than MELD baseline of <20 group with a significant statistical difference after MSCT (P = 0.003).  Nine articles (D value of MELD. The early assessment of the efficacy of MSCT could be based on variations of ALT at half a month and TBIL at 2 and 3 months. And it had beneficial effects for patients with LF, especially in ACLF based on the  Liver failure (LF) is defined as decompensation complications performing ascites, encephalopathy, and coagulopathy of any degree, and other physiological function of liver is damaged  , 2. In tHelicobacter pylori had a risk of carcinogenesis after MSC intervention  + 3.78∗In [bilirubin (mg/dL)] + 11.2∗In (INR) + 6.43, which ranged from 6 (mild disease) to 40 (severe disease) [D value of MELD of 13.92 ± 2.27 compared with MELD baseline of <20 group (1.46 ± 2.18) after MSCT (P = 0.003)  . All inc= 0.003) . But bey= 0.003) .Only three studies are involved in the survival of LF patients in treatment of MSCT. Lin et al.  showed aMSC transplantation has been utilized gradually in clinical practice and been emerged as a novel intervention for the treatment of liver cell failure. And some articles demonstrated its advantages deeply and explored a systematic protocol , 42. Of The improvement of liver function was found after MSC treatment in a short time of less than 3 months, especially ALT , which might be closely linked with the mechanisms of MSCs in the treatment of patients with LF. Wang et al.  hypothesMELD score was an objective assessment of patients with liver disease and was calculated by using a combination of blood tests: creatinine, serum bilirubin, and INR, whereas it lacks the information of portal hypertension . And theThere were several limitations. Firstly, the number of cases included in this review is small and the published works may not have covered all relevant references. Secondly, we are lack of the overall data of the type of cell—adipose-derived MSC, umbilical cord-derived MSC, and bone marrow-derived MSC; we thus could not compare their differences in treatment of liver diseases. Thirdly, there are two studies enrolled of less than five cases and evidence might be weak. Finally, almost no information on clinical symptoms  was provided in the studies we included.Our study analyzed the improvement of liver functions  after MSCT and the impact of MSCT on MELD score characterized by immune tolerance of stem cells . All of


This study aimed to establish reference ranges of bone markers in Chinese adolescents between the age of 12 and 16, and to search these markers’ characteristics and influential factors. Personal information and fasting blood samples were collected from 174 healthy adolescents in Northeast China. Serum levels of PINP, ALP, β-CrossLaps, calcium, phosphate, PTH, 25(OH)D and TSH were measured. Reference ranges were established for PINP [ng/ml], ALP [(53.88–463.63)U/L], β-CrossLaps [(0.16–1.19)ng/ml], calcium [(2.35–2.70)mmol/L], phosphate [(1.17–2.06)mmol/L] and PTH [(2.64–43.36)μg/L] in this population. We observed that bone formation markers PINP and ALP levels were evidently higher when compared to kit references for adults. Reference ranges for calcium, phosphate and PTH also differed from those provided by kit manuals. Serum ALP, PINP, phosphate and PTH changed with age , and were significantly higher in boys than in girls . Serum 25(OH)D and TSH levels didn’t correlate with PINP, ALP and β-CrossLaps . In conclusion, unique reference ranges should be provided for adolescents. BMI, sex and age independently influence certain serum bone markers in adolescents. Vitamin D deficiency is widespread. Serum levels of vitamin D and TSH may not influence bone turnover markers in this population. Their measurements can be useful in the assessment of metabolic bone diseases and growth disorder in adolescents. There are a number of common markers used in evaluating bone metabolism. Some of these markers are known as bone formation markers, including bone-specific alkaline phosphatase (BSAP) and procollagen type I N-terminal propertied (PINP). Another group of markers are known as bone resorption markers, such as β-CrossLaps, which is the most sensitive one. Bone mineralization is dependent on adequate intakes of calcium and phosphate, which can be reflected by measuring serum calcium and phosphate levels. Parathyroid hormone (PTH) adjusts the concentration of ionic calcium in blood and is important in bone remodeling. Vitamin D plays a key role in calcium and phosphate metabolism and it is essential for bone health2. The hypothalamic–pituitary–thyroid axis plays an key role in skeletal development, acquisition of peak bone mass and regulation of adult bone turnover3. Thyroid stimulating hormone (TSH) produced from the axis is a sensitive index of the thyroid function, and has been proposed to be a direct negative regulator of bone turnover in adult4. As for our concern, there is limiting research regarding the exact relationship between adolescents’ bone markers and TSH levels.In addition, thyroid function are also important in bone maintenance5, which have not been fully elucidated. These arose our interests in further investigation.It should also be noted that bone metabolism in adolescents differs from that of adults. New bones are growing and remodeling faster with growing age, due to high skeletal growth velocity and rapid bone turnover in adolescents. Bone marker references in this specific population may have unique characteristic compared with those of adultsIn the present study, we measured bone markers in a group of 12 to 16 years old adolescents in Northeast China, aiming to understand the reference ranges of bone markers such as serum levels of PINP, β-CrossLaps, alkaline phosphatase , calcium, phosphate and PTH, in this age group. In addition to age and sex, we examined nutritional status in terms of body mass index (BMI), 25(OH)D and TSH levels for any correlation between these variables and bone markers levels.The general distributions of BMI, bone markers, 25(OH)D and TSH in 174 subjects (82 boys and 92 girls) are shown in Fig. th and 97.5th percentiles of serum concentrations. Shown in Table Reference ranges of bone markers in this adolescent population were defined as the central 95%, between 2.5From our results Table , bone ma6 D and TSH were analyzed in correlation with bone markers in adolescents. Results are shown in Table Multiple regression analysis was used to identify covariates that might influence bone turnover marker values. The covariates included BMI, age, sex, 25(OH)D and TSH. Results are shown in Table In this study, we established serum bone marker references within healthy Chinese adolescents between the aged of 12 to 16. These markers included bone formation markers (PINP), bone resorption marker (β-CrossLaps), ALP, bone related mineralization markers  and PTH. In fact, compared with BSAP, ALP is not a sensitive and specific parameter to reflect bone metabolism, because ALP includes not only BSAP but also liver alkaline phosphatase. Nonetheless, when liver alkaline phosphatase is stable and remains within normal value, ALP presents the same trend as BSAP. Given the fact that ALP measurement is easier to obtain, cost-effective and more commonly used in China, it was examined and analyzed in the present study. For PINP, β-CrossLaps and PTH, kit manuals do not provide specific reference ranges for these markers in adolescents, neither do they mention the applicability of kit reference ranges to adolescent population. For ALP, calcium and phosphate, the kit manuals recommend reference ranges for both adults and adolescents, however, these ranges were not evaluated by the kit for they were quoted from other publications. We found that in our adolescent population, reference ranges of serum PINP and ALP were significantly higher than the kit’s reference for adults, and reference ranges of calcium, phosphate and PTH also differed from those provided by the kit’s manufacturer. As a result, reference ranges of most bone markers proposed in kit manuals are not suitable for evaluating adolescents. The present study suggests the necessity of establishing unique reference intervals for healthy adolescents in laboratories.7. According to previous publications, growth spurt occurs approximately between the age from 10 to 12 in girls and 12 to 14 in boys8. After the period of accelerated growth, biochemical markers of bone remodeling decrease but boys marker remain higher than in age-matched girls9.Of all the factors that may influence bone markers in adolescents, we found that sex difference played a role in bone marker concentrations. Serum ALP, PINP, β-CrossLaps, phosphate and PTH levels in boys were higher than the levels in girls, suggesting a strong relation of these markers to the later male pubertal growth spurt, as has been shown by others10. Age is another important variable that affects concentrations of bone formation markers in adolescents11. There are two peaks of bone growth during childhood development. The first peak appears during infancy; the second peak occurs during early puberty. Recent study found that bone markers increased rapidly during early puberty when growth velocity was highest and the period of most rapid bone mineral accrual13. Being indexes in the association with rapid growth velocities in healthy people, decreases in serum ALP and PINP as individuals growing older after early puberty come as no surprise.This study also showed that serum levels of the bone markers ALP and PINP were the highest in age 12–13 and significantly decreased with increased age in adolescents. This results are in accordance with previous reports14. Based on this criteria, the phenomenon of widespread vitamin D deficiency among Northeast Chinese adolescents has been observed in our study, with majority of recorded 25(OH)D levels being under 20 ng/ml (50 nmol/L). This result can not represent the entire Chinese adolescent population, because we only recorded 25(OH)D levels from adolescents aged from 12 to 16 in Northeast China, but similar findings have been demonstrated in another study which concluded that Chinese children tend to be vitamin D deficient15. As previously reported, lack of outdoor activities and inadequate sunshine exposure are main reasons caused most low level of 25(OH)D16. Usages of sunscreen, the short sunshine duration in the north of China and lack of purchasing channels of vitamin supplements are also notable predictors of vitamin D deficiency17. These may explain the results obtained from the present study. The phenomenon of widespread vitamin D deficiency among adolescents worldwide18 makes it difficult to find a perfectly normal population, in which sufficient nutrition status is achieved, for building up reference ranges for bone markers. However, it should be noted that controversies remain in how to define the “reference range” of vitamin D, and remain in whether the criteria of hypovitaminosis D proposed by the Endocrine Society is applicable to all population19. In the present study, following the method used in previous study20, we did not include results from subjects with 25(OH)D < 10 ng/ml in final analysis, in order to avoid marked impacts that may be caused by severe vitamin D deficiency. Excluding these subjects did not significantly change the results.Serum 25(OH)D is an index evaluating vitamin D nutrition status. According to the Endocrine Society’s guideline, vitamin D deficiency is defined as 25(OH)D below 20 ng/ml (50 nmol/L), vitamin D insufficiency is defined as 25(OH)D between 21 and 29 ng/ml (52.5–72.5 nmol/L), and vitamin D sufficiency is defined as 25(OH)D above 30 ng/ml (75 nmol/L). Thus, hypovitaminosis D is defined as 25(OH)D below 30 ng/ml (75 nmol/L)21. By promoting calcium and phosphate absorption in intestines, as well as increasing osteoclast numbers and stimulating bone resorption, vitamin D and PTH work together to maintain serum calcium and phosphate relatively stable, which is very important for bone development in adolescents22. However, it should be noted that serum 25(OH)D measurements are not suitable for bone turnover follow-up in this particular adolescent population, not only because of the high prevalence of vitamin D insufficient, but also due to lack of correlation between 25(OH)D and bone turnover markers.Our analysis suggested that serum 25(OH)D level was weakly negatively correlated to PTH level, but had no correlation with calcium and phosphate. This finding is in accordance with the well-adapted knowledge that vitamin D is not the only regulatory factor for calcium and phosphorus metabolism. Vitamin D co-acts with PTH hormone to be involved in the complex bone-kidney-parathyroid loop23, few studies proved the effects of thyroid hormones and TSH on bones in terms of bone markers. Normal level of thyroid hormone is important in keeping bone health, even subclinical hyperthyroidism, defined by a suppressed TSH level in the presence of normal thyroid hormone concentrations, is associated with fracture24. TSH receptor (TSHR) has been demonstrated in both osteoblasts and osteoclasts, suggesting TSH may exert direct action in these cells4. Thus, we suspected thyroid function may affect bone markers in adolescents. However, in our study, we did not find the correlation we expected. Further researches on this topic will benefit from a larger sample size.Finally, our study showed no correlation between serum TSH levels and bone markers in healthy adolescents. Previous studies only demonstrated that low-normal TSH values were associated with an increased prevalence of vertebral fractures in post-menopausal womenSome limitations do exist, despite the fact that present study has provided relatively thorough information on bone markers in adolescents. First of all, BSAP, instead of total ALP, should be detected as a more sensitive bone formation marker. Secondly, we did not obtain data on Tanner staging, which helps to evaluate puberty in adolescents, thus we could not accurately assess the association between early puberty and bone markers in the studied population. Thirdly, in view of our current results, to establish accurate age and sex-specific reference ranges in adolescents should be considered. Unfortunately, our study could not reach this goal, because subject number of each age and sex-specific group was not big enough to set up a reference range. Lastly, we did not collect information on outdoor activities and sunshine exposure of these adolescents, so we could not elucidate real reasons for their vitamin D deficiency. These limitations need to be improved in future in-depth studies.In conclusion, we have established reference ranges for bone markers PINP [ng/ml], ALP [(53.88–463.63)U/L], β-CrossLaps [(0.16–1.19)ng/ml], calcium [(2.35–2.70)mmol/L], phosphate [(1.17–2.06) mmol/L] and PTH [(2.64–43.36)μg/L] in an adolescent population from Northeast China. References provided by kits can not apply to this specific population. BMI, sex and age independently influence certain serum bone markers in adolescents. Vitamin D deficiency is widespread. Serum levels of vitamin D showed weakly negative correlation to PTH, but along with serum TSH, may not affect bone turnover markers in this population.25. Demographic information and medical history, including sex, age, previous medications intake, as well as vitamins and other supplements intakes were collected from each subject. The final analysis excluded 22 subjects, including one who suffered a bone fracture within 6 months; two who took vitamin D and calcium supplements within 3 months; two with history of autoimmune thyroid diseases; one with type 1 diabetes; and sixteen with serum 25(OH)D < 10ng/ml, due to the concern that severe vitamin D deficiency would affect reference ranges of bone markers20. Results from a total of 174 adolescents (82 boys and 92 girls) were eventually reported. Height and weight were measured during recruitment between December 2010 and March 2011, and body mass index (BMI) was calculated as an indicator for nutritional status.One thousand, three hundred and twelve students from middle schools were recruited for the previous epidemiological investigation. The investigation took place in Anshan, Northeast China. Using stratified cluster-sampling method, we initially selected 196 adolescents from them for the present study. The target population was aged from 12 to 16 years oldFasting blood samples (5–10 ml) were collected from each subject between 7:00am and 9:00am to minimize the effects of the diurnal variation of bone remodeling markers. Serum separations were completed within 2 hours upon collection (3000r/10 min). Aliquots of serum were stored at −80 °C until tests were run. Sample collections and separations were performed according to standard laboratory procedures.Serum PINP, β-CrossLaps, 25(OH)D, PTH and TSH levels were determined by electrochemiluminescence immunoassays (ECLA) using Roche commercial kits . Serum ALP, calcium and phosphate levels were determined using the Roche P-modular automated analyzer . All measures were done within two days in June, 2014. Intra- and inter-assay coefficients of variation of all the commercial kits were < 10%.26. P value of 0.05 was used otherwise. Reference ranges of serum bone markers were defined as the central 95%, ranged between the 2.5th and 97.5th percentiles of their concentrations20.Data were analyzed using SPSS software . Parametric data were reported as mean ± SD. Nonparametric data were reported as median with interquartile range. Bone marker concentrations were tested for their normal distribution. Logarithmic or square root transformations were applied to variables as needed to achieve a distribution as close as possible to normal. The parametric variables were compared using t-test between two groups and Kruskal-Wallis one-way ANOVA test among multiple groups, respectively. Pearson and Spearman correlation coefficients were used to find correlation between parametric and nonparametric variables, respectively. P level of 0.005 was considered significant for multiple comparisons among 5 groups when necessaryEthical approval was obtained from the Research Ethics Committee of Shengjing Hospital of China Medical University and methods were conducted in accordance with approved guidelines and regulations. All subjects and subject’s parents or guardians from the cohort provided his or her written informed consents for using their data and blood samples.The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.


Menopause is associated with a decrease in the level of sex hormones such as ovarian estradiol and progesterone and can cause various symptoms such as depression, hot flash, fatigue, heart palpitations, and headache. Furthermore, there is a risk of developing complications such as osteoporosis, cardiovascular diseases, Alzheimer's disease, and ovarian cancer. Schisandrae Fructus (SF) is widely used in Korean medicine as a cure for such complications. This study was conducted to evaluate the therapeutic effects of SF against menopause symptoms associated with follicle depletion caused by the industrial chemical 4-vinylcyclohexene diepoxide (VCD) in mice. VCD directly targets the preantral follicles. Mice were injected with VCD  daily for 15 days and then with SF dosage 3 times/week for six weeks. To evaluate the effects of SF, body weight, tail skin temperature, uterine weight, lipid profile, and osteocalcin levels were measured. A decrease in body weight and tail skin temperature and an increase in uterine weight were observed upon SF treatment. Moreover, SF treatment significantly decreased total cholesterol, triglyceride, osteocalcin, and low-density lipoprotein levels and low-density/high-density lipoprotein ratio. These results suggest the potential use of SF in the treatment of menopausal symptoms in women. Menopause is a natural process experienced by women . It is cIn a previous study, an ovariectomy model (OVX) and a chemical model [4-vinylcyclohexene diepoxide (VCD)] were used to study menopause in women . The OVXSchisandrae Fructus (SF), which belongs to the Magnoliaceae family, is well known in Korean medicine. SF comprises sweet (fruit skin), sour (pulp), bitter/pungent (seed core), and salty components  , 19. Accβ-estradiol  were purchased from Sigma-Aldrich Korea . VCD was dissolved in sesame oil  and E2 in EtOH (0.5 mg/kg).VCD (94956) and 17The extract of SF was provided from Biomix . A decoction of the traditional medicine was prepared by extracting it with 30% EtOH for 3 h. Moreover, the remains of the herbs and impurities were separated from the extracted liquid by a filtration separation process. After filtration and drying, extracts of SF were remained liquefied. All extracting processes of it were controlled by Biomix Inc. SF was carefully measured to prepare the necessary doses for each treatment group and then dissolved in distilled water for 30 min at a controlled room temperature. SF was orally administered to mice at 25, 100, and 400 mg/kg/day for 6 weeks.μm syringe filter, and the filtrate was used as the test solution. Ten microliters of the test solution was injected into an HPLC system . The analysis involved an isocratic elution of acetonitrile and 0.1% formic acid  over 40 min at a flow rate of 1 ml/min.Gomisin N was purchased from the National Development Institute of Korean Medicine. HPLC-grade acetonitrile was purchased from Daejung Inc. The compound structure is presented in n = case) and allowed to acclimatize (object) for 1 week before treatment with VCD. Mice were randomly divided into five experimental groups (n = 8/group): negative control mice received an intraperitoneal (i.p.) injection of sesame oil daily for 15 days. VCD + phosphate-buffered saline (PBS) group, VCD + 17β-Estradiol (E2) group, and VCD + SF-treated groups : mice received an i.p. injection daily of VCD (160 mg/kg) for 15 days. PBS was used as a vehicle control for SF. After dosing with VCD, PBS and each treatment were orally administered thrice a week during 6 weeks. Two days after the last dose (day 65), mice were sacrificed by CO2 asphyxiation, and uterus and serum samples were collected for analyses. No mice died during the experiment. All the experiments and animal handling procedures in this study were approved by the Animal Experimental Ethics Committee of the Kyung Hee University [KHUASP (SE)-16-037].Six-week-old C57BL/6 female mice were purchased from Orient Bio . The mice were group-housed . Three measurements were obtained 10 min apart, and the average value was used as a single data point.μL per well). Optical density of the samples was measured at 450 nm using a microplate reader .Fasting serum samples were collected and stored at −80°C before analysis. The osteocalcin levels in the serum samples were determined using a commercial enzyme immunoassay kit  according to the manufacturer's instructions. A 96-well microtiter plate was coated with an anti-mouse OC monoclonal antibody. The standard and sample were then added and incubated for 1 hat room temperature. A second ary peroxidase-labeled anti-Gla-OC monoclonal antibody was then added and incubated for 1 h. Finally, the plates were treated with the substrate solution (TMBZ) for 30 min, and the reaction was stopped with the addition of the TMBZ stop solution . The serum biochemistry parameters, including total cholesterol, triglyceride, low-density lipoprotein (LDL), and high-density lipoprotein (HDL), were analyzed.For serum biochemical analysis, the clotted blood samples were centrifuged for 10 min at 1,500p value of <0.05 was considered statistically significant.Statistical analysis of the data was conducted using Prism 5 software . All the values are presented as mean ± standard error of the mean (SEM). Statistical significance was determined by one-way ANOVA, followed by Tukey's test using randomly selected samples. A μg/ml). Linearity was evaluated using the correlation coefficient (R2) of the calibration curve. Gomisin N was identified at a retention time of 25.085 min and its UV spectrum is shown in μg/g.SF was characterized using Gomisin N by HPLC analysis . By consOn day 65, we collected serum samples from all groups to investigate the lipid profile. Total cholesterol, LDL, and LDL/HDL levels were significantly higher in the VCD + Vehicle group than in the control mice Figures . TriglycWe observed body weight and tail skin temperature for 9 weeks. After dosing (day 15), a gradual difference in body weight and tail skin temperature was observed in control mice and VCD-treated animals. From the eighth week, body weight of mice in the VCD group was significantly higher than that of control group mice . Mice inPrevious studies have demonstrated that osteoporosis is associated with menopause. After menopause, osteocalcin levels increase owing to the lack of sex hormones such as estrogen and androgen. Furthermore, osteocalcin is considered a bone turnover marker , 25. Theβ-estradiol level and estrous cyclicity [Menopause is recognized by the cessation of menstrual cyclicity for at least one year , 27. Theyclicity . Accordiyclicity , 14, VCDyclicity . TherefoIn clinics, HRT is widely used for the treatment of menopause-inducing symptoms. This therapy can alleviate menopausal symptoms and protect women from diseases such as osteoporosis and cardiovascular diseases , 39. AccIn many Asian countries, SF (Magnoliaceae family) is widely used in traditional medicine for the treatment of menopausal symptoms. SF is known as wu-wei-zi in Chinese , GomishiOsteoporosis, a disease with decreased bone strength and increase in the risk of a broken bone, generally develops after menopause in postmenopausal women , 65. SomOur data suggest that SF can successfully ameliorate menopausal symptoms in VCD-induced ovarian failure mice. SF could be used as a potential candidate for ameliorating symptoms of menopause in women.


A small percentage of people with autism spectrum disorders (ASD) have alterations in chromosome 15q11.2-q3, the critical region for Prader-Willi syndrome (PWS). Data are limited, however, on the rates and characteristics of ASD in PWS. Previous estimates of ASD in PWS (25 to 41%) are questionable as they are based solely on autism screeners given to parents. Inaccurate diagnoses of ASD in PWS can mislead intervention and future research.M = 11) were assessed with the Autism Diagnostic Observation Schedule-2 (ADOS-2). An expert clinical team-made best-estimate ASD diagnoses based on ADOS-2 videotapes, calibrated severity scores, and children’s developmental histories and indices of current functioning. Children were also administered the Kaufman Brief Intelligence Test-2, and parents completed the Repetitive Behavior Scale-Revised and Vineland Adaptive Behavior Scales. Scores were compared across children with PWS + ASD versus PWS only. The performance of an ASD screener, the Social Communication Questionnaire (SCQ) and the ADOS-2 were evaluated in relation to best-estimate diagnoses.One hundred forty-six children and youth with PWS aged 4 to 21 years  PWS genetic subtype. Compared to the PWS-only group, children with PWS + ASD had lower verbal and composite IQ’s and adaptive daily living and socialization skills, as well as elevated stereotypies and restricted interests. Regardless of ASD status, compulsivity and insistence on sameness in routines or events were seen in 76–100% of children and were robustly correlated with lower adaptive functioning. The SCQ yielded a 29–49% chance that screen-positive cases will indeed have ASD. The ADOS-2 had higher sensitivity, specificity and predictive values. Communication problems were seen in children who were ADOS-2 positive but deemed not to have ASD by the clinical team.Autism screeners should not be the sole index of probable ASD in PWS; children need to be directly observed and evaluated. Compulsivity and insistence on sameness are salient in PWS and likely impede adaptive functioning. Most children with PWS only evidenced sub-threshold problems in social interactions that could signal risks for other psychopathologies.The online version of this article (doi:10.1186/s11689-017-9200-2) contains supplementary material, which is available to authorized users. Prader-Willi syndrome is a neurodevelopmental disorder that results in a complex behavioral and developmental phenotype. Caused by a lack of paternally derived imprinted genes on chromosome 15q11-q13, people with Prader-Willi syndrome (PWS) typically manifest mild to moderate intellectual disability, compulsivity, rigidity, irritability, social dysfunction, growth hormone deficiencies, and hyperphagia that can lead to life-threatening obesity . ApproxiMAGEL2 [Individuals with mUPD (versus deletions) are at higher risk for autism symptoms or autism spectrum disorder (ASD). They are also at heightened risk for psychotic illness, often with a depressive or affective component, which typically begins in adolescence or young adulthood , 5. HighMAGEL2  and the MAGEL2 , were idAlthough disruptions in the PWS 15q11-q13 region are thus often implicated in ASD, data are relatively limited on rates of ASD in PWS. Studies to date are of some concern, as most have relied on autism screeners that are completed by parents, and have not directly observed or evaluated offspring with PWS. Autism screeners identify children in need of further evaluation and are not meant to be diagnostic. Even so, such tools are widely used in research to index probable ASD. Veltman et al. ) to pareA second complication involves the salience of repetitive behaviors in the PWS phenotype. Clinically, we find that families, educators, and other professionals often use these easily observed behaviors to raise suspicions of co-occurring ASD in PWS, even though such behaviors alone are not diagnostic of ASD. Repetitive behaviors in PWS are indeed highly reminiscent of those seen in ASD, including insistence on sameness, repetitive questioning or speech, and ordering and arranging items –19.Comparing children with PWS versus ASD on the Childhood Routines Inventory , GraevesThe current study addresses these methodological concerns and also provides a more complete characterization of children with PWS and ASD compared to those with PWS only. We administered the Autism Diagnostic Observation Schedule , and thn = 8) or imprinting defects (n = 7).The sample included 146 children and adolescents aged 4 to 21 years with genetically confirmed PWS. Children and families were recruited from across the country for an ongoing study on behavior and development in children and adults with PWS. Children were averaged 11.4 years of age and were evenly distributed across gender . Regarding genetic subtypes of PWS, 52% had paternal deletions , and 37.7% had mUPD. Fifteen children (10.3%) had either atypical deletions  is a win = 128) were administered Module 3 for verbally fluent children, while 18 were administered Module 2, designed for those who are less fluent and use phrase speech. Compared to children receiving Module 3, those administered Module 2 were younger  = −4.74, p < 0.001), but did not otherwise differ on cognitive or behavioral measures.The majority of participants in this study (n = 25). We considered Module 4 (geared for adolescents and adults) and piloted it with four adults with PWS aged 24 to 30 years who were not included in the present study. These adults struggled with the more abstract items and the conversational emphasis of Module 4. ADOS-2 test guidelines note that clinicians need to determine module fit based not only on age or language skills but also on the relevance of tasks to the examinee’s interests and abilities. Instructions further note that, when in doubt, examiners should use modules that are well within reach of the examinee’s language skills. We thus opted to use Module 3 in 17–21-year-old study participants.Although ADOS-2 calibrated scores are pertinent for Modules 1 to 3 in children up to age 16 years, we also administered Module 3 to youths aged 17 to 21 years   is a 40-A demographic questionnaire identified participant’s psychotropic medications, growth hormone treatment, age, gender, PWS genetic subtype, body mass index, family income, and parental education. These were used as correlates of ASD status and symptoms. We interviewed parents to obtain children’s developmental and medical histories, which also included a family medical history. These data assisted with the best-estimate diagnoses of ASD.M = 100, SD = 15) for a verbal, nonverbal, and overall IQ composite score. It has excellent psychometric features, including test-retest reliability of the composite (r = 0.90), verbal (r = 0.91), and nonverbal IQ scores (r = 0.83).Participants were individually administered the Kaufman Brief Intelligence Test-2  which wM = 100; SD = 15).The Vineland Adaptive Behavior Scales-2 Survey Form  is a wiThe Repetitive Behavior Scale-Revised (RBS-R) assesses a wide range of restricted and repetitive behaviors in people with developmental disabilities . InformaBased only on ADOS-2 Calibrated Severity Scores, 32 children (21.9%) were classified as having ASD. After the clinical team reviewed ADOS-2 data and videotapes, along with children’s medical and developmental histories and current functioning, the rate was lowered to 18 children, or 12.3% of the sample. We used these second clinically informed ASD determinations in subsequent data analyses.M = 65.80, SD = 13.87) relative to both the PWS-only  and PWS + ASD groups ; F(2144) = 7.14, p < 0.001.Follow-up analyses explored possible differences between children with or without ASD, and the 14 children who were positive on the ADOS-2 yet were judged clinically to not have ASD. These 14 children did not differ from their peers in age, gender, BMI, or genetic subtype. As summarized in Additional file X2 (2) = 6.11, p < 0.05. Although based on parent descriptions only, no differences were found in specific problems with articulation or fluency, seen in 75 and 18.6% of the sample, respectively. However, difficulties getting thoughts into words or verbal apraxia were more frequently reported in the ADOS-2 positive, clinically negative group (76.9%) compared to those with PWS only (20.4%) or PWS + ASD (16.7%), X2 (4) = 15.82, p < 0.01. No group differences were found in speech/language therapy, with 68% of participants receiving these services.Based on this finding, we explored three speech-related items from participants’ developmental histories: (I) Does your child have speech/language problems? (2) If so, please describe. (3) Does your child receive speech/language therapy? Children who were ADOS-2 positive but clinically negative were more likely to have speech problems (100%) than those with PWS + ASD (64.7%) or PWS only (78.4%); n = 13; 72.2%) than girls ; X2 (1) = 4.57, p < 0.05). Participants with the mUPD subtype were more likely than their counterparts to receive an ASD diagnosis, X2(3) = 13.31, p < 0.01. Of the 18 children with PWS + ASD, 14 had the mUPD subtype, 2 had imprinting defects, and 2 had type II deletions.ASD diagnostic status was not significantly correlated with child age; these diagnoses were identified in 10 children and 8 adolescents. Children with versus without ASD both averaged 11 years of age  and with social affect severity scores . IQ scores were not associated with the ADOS-2 repetitive behavior domain (r’s range −0.10 to −0.11). A similar pattern emerged in the Vineland’s communication, daily living skills, and socialization domains with overall severity scores  and the social affect domain , but not the repetitive behavior domain. No other significant correlations were found.For the sample as a whole, KBIT-2 verbal, nonverbal, and composite IQ scores were negatively correlated with ADOS-2 overall calibrated severity scores  = −0.48, p < 0.001; PWS + ASD r(14) = −0.54, p < 0.05). Remaining ADOS-2 items were elevated in those with PWS + ASD, but low in the PWS-only group.As expected, social and communicative impairments were highly prevalent in the PWS + ASD group. Because social dysfunction is often seen in people with PWS, we wondered to what extent the PWS-only group manifested problematic social and communication scores on the ADOS-2. Figure t tests of RBS-R domains revealed that the PWS + ASD group had significantly higher scores in restricted behavior, stereotypies, and RBS-R total scores. While they also scored higher on the compulsive and sameness/rituals domains, these differences were not as robust (p’s <0.05). No PWS group differences emerged in the self-injurious behavior domain, which in PWS is primarily manifest as skin picking.As shown in Table r’s = −0.24 and −0.22, respectively, p’s <0.01). Compulsivity and insistence on sameness domains were negatively correlated with the all three Vineland domains: communication , daily living skills , and socialization domains . No other significant correlations were found.RBS-R scores were not significantly associated with age, gender, PWS genetic subtypes, and psychotropic medication or growth hormone treatment. Nonverbal and composite IQ scores were negatively correlated with the stereotypies domain  = −5.13, p < 0.001.)Based on the SCQ cut-point of ≥15, 32.87% of the sample was classified as having a possible ASD. Not surprisingly, SCQ total scores were higher in those with PWS + ASD, The test sensitivity of the SCQ, 77.78%, indicates that ASDs were accurately detected by the SCQ in the majority of participants with PWS + ASD  of 29.17%, suggesting a very low probability that a child with PWS who screens positive on the SCQ will indeed have an ASD.PPV’s vary greatly depending on the overall prevalence of a disease or condition, and as such, a lower PPV and higher NPV are not unusual given the low rate of PWS + ASD (12.3%) in our sample. We thus calculated an adjusted PPV based on the higher estimates of PWS + ASD noted in the literature of ~25% , 16, usiThe test sensitivity of the ADOS-2 was 1.00, indicating that it accurately detected all cases of children with PWS + ASD; none were missed. The test specificity of 89.06% suggests that the ADOS-2 also ruled out ASD in the majority of children who did not receive ASD diagnoses. Further, the NPV of 1.0 suggests a high probability that children with PWS who fall below ADOS-2 thresholds will not have an ASD diagnosis  sensitivity and specificity analyses are not recommended. Instead, we calculated Cohen’s kappa and the percentage of positive agreement (PPA) and percentage of negative agreement (PNA) . AdditioThe PPA and NPA index agreement between measures, not if they are correct in their agreements or disagreements. As such, we compared agreements and disagreements to ASD diagnostic status. As Table This is the first large-scale study to identify the rates and characteristics of ASD in a large cohort of children and youth with PWS based on standardized autism assessments combined with thorough clinical reviews. Findings provide new insights into how children with PWS + ASD differ from those with PWS only in cognitive and adaptive functioning, repetitive and social behaviors, gender, and PWS genetic subtypes. Further, by determining how well the SCQ screener and ADOS-2 performed in relation to ASD diagnoses, we generate new recommendations for future research.Using best-estimate ASD diagnoses, our 12.3% rate of ASD in 146 children with PWS falls below the 25 to 41% reported in previous studies , 16. SevSecond, the clinical team may have been too conservative in making ASD diagnoses, as reflected in the 14 children who were positive on the ADOS-2 but were judged by the team to not have ASD. It is important to emphasize that the above threshold scores on the ADOS-2 are not a substitute for a diagnosis of ASD. Instead, this observational schedule provides valuable data for experienced clinicians to use in their diagnostic formulations, along with children’s developmental and medical histories and current cognitive, adaptive, and behavioral functioning .both the PWS-only and PWS + ASD group on the Vineland’s communication domain. Following up this finding with developmental history data, the ADOS-2-positive, clinically negative group had higher rates of speech problems than their peers, especially in verbal apraxia or difficulties getting thoughts into words. Anecdotally, the research team observed that these children typically had reciprocal social intentions, but their struggles to communicate diminished the quality of their interactions. Because we did not obtain external confirmation of parental reports of verbal apraxia, or quantify the team’s clinical impressions, these results should be interpreted cautiously. Even so, socially reciprocal intentions in children with diminished communication skills offer a reasonable explanation for why these children did not receive ASD diagnoses. Our analyses of this group also aptly demonstrate the value of thorough clinical reviews of ADOS-2 and other pertinent child data in formulating ASD diagnoses.The importance of this multi-modal evaluative process is highlighted by analyses of the 14 children who were ADOS-2 positive but clinically negative. Although these children had, on average, similar cognitive and adaptive behavior scores as the PWS + ASD group, they scored significantly lower than M = 86.88, SD = 14.09, range 72–112). A second possibility is that the team was more inclined to make ASD diagnoses in lower functioning children. However, the team’s evaluation of 14 children who were ADOS-2 positive but clinically negative argues against this possibility. These 14 children had relatively low IQ’s , and 80% of them had IQ’s <70. Despite reaching ASD threshold on the ADOS-2, this group of predominantly lower functioning children was judged not to have an ASD diagnosis. The team thus appeared to adequately discriminate ASD across the IQ spectrum.A final possible bias is that children’s levels of cognitive functioning may have overly influenced the team. This is particularly relevant as children with PWS + ASD versus PWS only had, on average, lower IQ and adaptive behavior standard scores. Biases in relation to IQ could go in two directions. First, it is possible that the team simply did not recognize ASD in children who were higher functioning. This possibility, however, is offset by the fact that 33% of the PWS + ASD group had KBIT-2 composite IQs of 70 or higher . ThThe performances of the SCQ and ADOS-2 underscore the importance of combining ASD evaluation tools with thorough clinical reviews. With the SCQ’s low positive predictive value (PPV), there was just a 29% chance (49% using the adjusted PPV) that children with PWS who screen positive will indeed have ASD. Both of these PPVs fall at or below chance levels. The ADOS-2 performed much better, with enhanced specificity and sensitivity, but it too yielded a PPV just above chance (56%), which improved to 75% using the adjusted PPV. Comparing agreement across the two measures, they did a better job agreeing on the absence versus the presence of ASD (PNP = 76.8% versus PPA = 58.8%). However, we also determined if the measures agreed or disagreed correctly, i.e., in relation to ASD diagnoses. The SCQ and ADOS-2 correctly agreed on children’s ASD status 67.8% of the time, far more than they agreed but made the wrong call (4.8%). However, when the measures disagreed, the SCQ made more wrong calls (22.6%) than the ADOS-2 (4.8%), and most of these were false positives.On the one hand, the high false positive rate of the SCQ indicates that the SCQ is performing as it was intended—to identify children in need of further evaluation. And in clinical or educational settings, a high false positive rate is often acceptable so that more children are evaluated and none are missed who could benefit from ASD interventions. Difficulties arise, however, in PWS research that only uses ASD screeners, without follow-up evaluations of screen-positive cases. Such practices risk creating a false impression that ASDs may be quite widespread in PWS.Our findings recommend two options for future PWS research. The fact that ADOS-2 and SCQ correctly agreed for 68% of participants suggests that the two instruments performed better together than at least the SCQ did alone. If researchers want to reduce error due to false positives, they should consider using agreements between at least two standardized indices of ASD and acknowledge that subsequent rates of ASD in PWS may be still inflated. If researchers aim to precisely identity ASD in PWS, then they need to use the ADOS-2 or other direct assessments of children. Further, these observations need to be placed in the broader context of children’s development, current functioning, and phenotypic features. Indeed, Hepburn and Moody  convinciThis study had several strengths, including a large, well-characterized sample and standardized, multi-modal ASD assessments that were reviewed by experts in ASD and PWS. Even so, several shortcomings need discussion. First, the study did not include a separate measure of language function and speech/language data relied solely on developmental history interviews with parents. Direct testing of language function would have been particularly helpful in characterizing the 14 ADOS-2-positive but clinically negative children. Most children with PWS have language delay, oral motor difficulties, poor articulation, slow rate of speech, flat intonation, and language skills that may fall below their level of cognitive functioning , 54. AltAnother limitation is that the study excluded adults with PWS. Given the scarcity of research on co-occurring ASD in adults with genetic neurodevelopmental syndromes, we opted to focus initially on children and youth with PWS. Recently, however, Sappok at al.  evaluateA final issue relates to the analyses of SCQ performance. Other researchers have used receiver operator curves to identify SCQ cut-points that provide optimal sensitivity or specificity for their study samples. We, however, used the recommended cut-point for this measure to enable comparisons to existing literature that used this same cut-point. We also reasoned that future PWS research would not necessarily benefit from a continued reliance on screeners, even with a revised PWS-derived cut-point.We recommend that multi-modal approaches and direct observations of children be used in future studies of ASD in PWS. Additional work is needed on gender differences in PWS + ASD, and the interplay between verbal apraxia or other language disorders and ASD diagnoses. Although individuals with the mUPD versus deletion subtype are at higher risk for ASD, it is unclear how ASD symptoms change over time, or if they relate to high risks for psychosis also seen mUPD cases. Regardless of ASD status, compulsive behaviors and insistence on sameness are salient in PWS and likely impede optimal adaptive functioning. In contrast, persistent stereotypies are unusual in PWS and when present in children may signal the need for further evaluation of their social communicative functioning and ASD status. Although social impairments fell below ASD thresholds in the majority of our sample, 45 to 70% with PWS only still evidenced some degree of impairment in the quality and amount of their reciprocal social interactions. These social impairments may be associated with other psychiatric, emotional, or behavioral disorders or simply be intrinsic to the PWS phenotype.


Aim. To evaluate the possible structural and functional changes in the retinal nerve fibre layer (RNFL) and the ganglion cell complex (GCC) of chronic smokers and compare them with those of passive healthy smokers using spectral domain optical coherence tomography (SD-OCT) and pattern electroretinogram (PERG). Materials and Methods. We include 80 active chronic smokers and 80 age- and sex-matched healthy passive smokers. After a full ophthalmological examination, SD-OCT and PERG were tested for all participants. Urinary levels of cotinine and creatinine with subsequent calculation of the cotinine creatinine ratio (CCR). Results. Inferior and superior quadrants of RNFL were thinner in group I, but nasal and temporal quadrants did not show significant difference between the groups. There were no significant differences of GCC values between the two groups. There was no significant difference of PERG-P50 amplitude and latency; however, PERG-N95 showed significant difference between the two groups. Multiple regression analyses demonstrated that the number of cigarettes/day, urinary cotinine, and PERG-N95 amplitude are the most important determinants for both superior and inferior RNFL thicknesses. Conclusion. RNFL thickness decreases in chronic, healthy, heavy cigarette smokers, and this thinning is related to the number of cigarettes/day, urinary cotinine, and PERG-N95 latency and amplitude. Several studies have suggested that cigarette smoking, which was proven to be a risk factor for atherosclerotic complications in the aortic, coronary, and cerebral circulation , causes Common eye diseases such as cataract , age-relGrzybowski and Holder  speculatPast studies reported the alteration in visual fields of healthy heavy cigarette smokers documented by decreased retinal sensitivity and peripheral scotomata in the visual fields . MoreoveThe peripapillary retinal nerve fibre layer (RNFL) and ganglion cell-inner plexiform layer complex (GCIPL) thickness, which were proved to be thinned in glaucomatous  and nongThe aim of this study was to evaluate the possible structural and functional changes in RNFL and GCC of chronic heavy smokers and compare it with those of passive healthy smokers using OCT and PERG.This cross-sectional study was conducted between January 2015 and May 2016, with the participants recruited from the patients' relatives and hospital staff of the outpatients' clinics of Ain Shams University Hospital. The study adhered to the tenets of the Declaration of Helsinki, and it was approved by the ethical committee of the Faculty of Medicine, Ain Shams University. Informed written consent was obtained from all individual participants before the study began.A total of 160 participants were enrolled in the study. The participants were classified into two groups; the active smoker group included 80 participants who were healthy cigarette smokers—who smoke at least 10 cigarettes for 10 years—with no other systemic or ocular disease (group I) and the passive smoker group consisted of 80 age- and gender-matched healthy never smoking participants but in close contact with smokers (group II). We could not add a 3rd group (never smoked and without exposure to passive smoking) as the smoking is highly prevalent in our environment. One eye of each participant was included.We included participants whose age ranged from 25 to 35 years, spherical refraction between +1.0 and −1.0 diopters, best corrected visual acuity (BCVA) of 20/20 or better, intraocular pressure (IOP) < 22 mmHg, and axial length (AL) < 25 mm. In group II, participants smoke at least 10 cigarettes/day for at least 10 years duration.We excluded any participant with pre-existing retinal diseases, glaucoma, previous LASIK or retinal surgery, previous eye trauma, a history of alcohol intake, any systemic disease specially diabetes mellitus (DM), uncontrolled hypertension, recurrent migrainous attacks, or a history of medication intake within the last 3 months, especially systemic vasoactive drugs.Full ophthalmological evaluations, including best corrected visual acuity (BCVA) using Snellen charts, slit-lamp biomicroscopy, Goldmann applanation tonometry, and fundus examination, were performed. Then one eye of each participant was subjected to the following investigations.Axial length measurement (AL) was peformed using A-scan ultrasonography . Prior to measurement, the cornea was anesthetized with one drop of topical 0.4% benoxinate HCl. Five readings were obtained to calculate an average value. The standard deviation was set below 0.1 mm for each subject.Central corneal thickness (CCC) measurement was performed using SD-OCT  by the anterior segment OCT program. We used the single corneal line scan horizontally with the scan width of 6 mm centered on the center of the cornea, in which the single scan was averaged from 10 A-scans to gain the best quality image and least noise. The resolution of the single scan of the 10 A-scans was 1024 points. Then the CCT was measured from the most superficial hyperreflective line representing the corneal epithelium to the deepest hyperreflective line indicating the corneal endothelium.Retinal nerve fibre layer (RNFL) measurement was performed using spectral domain optical coherence tomography (SD-OCT) . After pupillary dilatation using Mydriacyl 0.5% (Alcon Inc.) eye drops, the peripapillary RNFL was measured by 3 circular scannings around the optic nerve in an area with a diameter of 3.4 mm. We recorded the RNFL thicknesses at the superior, nasal, inferior, and temporal quadrants, and at each clock hour, the mean values were calculated. The RNFL thickness was differentiated from other retinal layers using edge detection algorithm.Ganglion cell layer (GCL) thickness map was performed with SD-OCT. The GCL thickness map was obtained from the macular map scan and calculated automatically by the device. It is composed of eight sectorial thickness measurements in two concentric circles with diameters of 4.5 and 9 mm centered on the fovea. The area bounded by the outer (9 mm) and middle (4.5 mm) circles forms the outer ring (OR), while the area bounded by the middle (4.5 mm) and inner (1.5 mm) circles forms the inner ring (IR). Each ring is divided into superior-temporal, superior-nasal, inferior-temporal, and inferior-nasal quadrants. STIM, SNIM, ITIM, and INIM stand for superior-temporal of the inner macula, superior-nasal of the inner macula, inferior-temporal of the inner macula, and inferior-nasal of the inner macula, respectively. STOM, SNOM, ITOM, and INOM stand for superior-temporal of the outer macula, superior-nasal of the outer macula, inferior-temporal of the outer macula, and inferior-nasal of the outer macula, respectively. The central 1.5 mm circle centered on the macula, being devoid of ganglion cells, has no measurement.Also, the central 9 mm circle centered on the fovea is divided into two halves: upper and lower halves with the total GCL thickness measured all over each half.Pattern electroretinogram (PERG) was recorded with the RETI-port/scan 21  using an implemented protocol in the system software. Binocular stimulation was used with suitable correction of any refractive error in relative with the eye-screen distance. No dilation of the pupils was required with the PERG to maximize the retinal image quality, and central fixation was determined (patient was monitored with a TV camera). Parameters of the PERG stimulation were the following: 21″ CRT monitor with a frame rate equal to 75 fps was used; black-and-white backward-moving checkerboard (30° field of view (FOV)) was exposed to the patient, with a check size equals to 1° 2′; temporal frequency of 4.0 ± 8.0 reversal rate (rps) is 2.0 ± 0.40 Hz; Michelson contrast is 97%; and luminance for white elements was 120 candelas per square meter (cd/m2).The electrodes placed were the following: the ground-gold disk electrode over the forehead, the HK-loop electrode into the lower fornix, and the surface reference electrodes on the skin near the ipsilateral outer canthus of each eye.μV)/division filters, filters of frequency is 1–100 Hz, artifact discard threshold was 95% (for the amplifier range ± 100 μV), notch filters were off, averaging of 200 sweeps, and sweep time was 250 ms (time base: 25 ms/division). Two successive waveforms were recorded; then, they were off-line being an average and interpreted. The PERG test parameters were in accordance with the ISCEV standard [Parameters of the recording system were as follows: amplifier sensitivity is 20 microvolts (standard .Urine samples were collected in aseptic plastic containers to measure urinary levels of cotinine  and creaUrinary cotinine level was determined by enzyme-linked immunosorbent assay (ELISA)  in the patients and control groups according to the manufacturer's instructions. A solid phase ELISA was employed based on the basic principle of a sandwich ELISA. The micro-ELISA strip plate wells are precoated with an antibody specific to cotinine. Samples are added to the wells and combined to the specific antibody. Then, a horseradish peroxidase- (HRP-) conjugated antibody specific for cotinine is added to each well and incubated. Free components are washed away. The TMB substrate solution is added to each well. Only those wells that contain cotinine and HRP-conjugated cotinine antibody will appear blue in color and then turn yellow after the addition of the stop solution. The optical density (OD) is measured spectrophotometrically at wavelength of 450 nm. The OD value is proportional to the concentration of cotinine.Urinary creatinine level was measured by the Jaffe kinetic reaction with picric acid .The cotinine creatinine ratio (CCR) of each sample was calculated from the equation CCR = urinary  cotinine  (ng/ml)/urinary  creatinine  (ng/mg).t-test and Pearson's correlation analysis were used for the statistical analysis of the data. Regression analyses were done to assess the different factors that can affect the RNFL thickness. Significance was evaluated at p < 0.05 levels.We analyzed data using the Statistica software, version 10. Quantitative variables were expressed as mean ± SD. Descriptive statistics were done using chi-square analysis. An independent sample The study included 80 participants (80 eyes) in each group; they were 75 males (93.75%) and 5 females (6.25%) in the active smoker group and 74 males (92.5%) and 6 females (7.50%) in the passive smoker group . Their aThere were no statistical significant differences between the two groups regarding age, visual acuity (VA), BCVA, spherical equivalent (SE), intraocular pressure (IOP), axial length (AL), and central corneal thickness (CCT) .The active smokers had smoked an average of 23.50 ± 9.69 cigarettes per day . The mean smoking duration of the active smokers was 19.05 ± 4.89 years  .t-test showed significant differences between the two groups for urinary cotinine (p < 0.0001) and CCR (p < 0.0001) (An independent  0.0001) .p < 0.0001 and p < 0.0001, resp.), but nasal and temporal quadrants did not show significant difference   .There were no significant differences of GCC values between the active smoker group and passive smoker group .There was no significant difference of PERG-P50 amplitude or latency; however, PERG-N95 amplitude and latency showed statistical significant difference between the active smoker group and passive smoker group .p < 0.0001) while showed positive correlation with PERG-N95 amplitude (p < 0.0001)  .R = 0.87, R2 = 0.75, adjusted R2 = 0.72, and F  = 21.195.Regression summary for superior RNFL thickness in the active smokers is R = 0.870, R2 = 0.760, adjusted R2 = 0.73, and F  = 21.97.Regression summary for inferior RNFL thickness in the active smokers is β = −0.85, −0.46, and 0.31 with p < 0.0001, <0.0001, and 0.02, resp.) and inferior RNFL thicknesses  (Tables Multiple regression analyses for the active smoker group show which factor in the study was the most important determinant of superior and inferior RNFL thicknesses. We found that the number of cigarettes/day, urinary cotinine, and PERG-N95 amplitude are the most important determinants for both superior  Tables  and 8.In the recent years, OCT and PERG have proven to be a reliable technology to assess the peripapillary RNFL and GCC , 16.We conducted this study to evaluate the effects of smoking on GCC and RNFL thickness. Our results showed that average RNFL and GCC were not affected. The analysis of the quadrants revealed that inferior and superior quadrants of RNFL were thinner in the active smokers than those of the passive smokers, but temporal and nasal quadrants were not. This deviates from the results reported by Moschos et al.  who founAlthough Dervişoğulları et al.  stated tThere was no significant difference of PERG-P50 amplitude or latency; however, PERG-N95 showed statistical significant difference between the active smoker group and passive smoker group. Worthy of notice is that PERG-N95 showed significant differences among active smoker and passive smoker groups which are the tests for optic nerve functions. These results might denote that smoking targets the RNFL and optic nerve.Although Hepsen and Evereklioglu  suggesteThe results obtained in our testing found that both superior and inferior RNFL thicknesses showed negative correlation with the number of cigarettes/day, urinary cotinine, and PERG-N95 latency while showed positive correlation with PERG-N95 amplitude.Multiple regression analyses were studied for the active smoker group to show which factor in the study was the most important determinant of superior and inferior RNFL thicknesses. We found that the number of cigarettes/day, urinary cotinine, and PERG-N95 amplitude are the most important determinants for both superior and inferior RNFL thicknesses.RNFL thickness decreased in chronic heavy smokers, and the thinning is related to the amount of smoking rather than duration; and it could be due to direct neurotoxic effect on the optic nerve and reduced blood flow due to the vasoconstrictive effect of nicotine .Yoshida et al.  suggesteThere was no correlation between duration of smoking and RNFL thickness. From these results, we concluded that the amount of smoking is more effective than the duration of smoking in RNFL thinning.The study limitations are the relatively small sample size, multicenter studies may be more valued, and cross-sectional design would not provide further and detailed results.In conclusion, our study results suggested that RNFL thickness decreases in chronic, healthy, heavy cigarette smokers, and this thinning is related to the number of cigarettes/day, urinary cotinine, and PERG-N95 latency while showed positive correlation with PERG-N95 amplitude.


Mast cells and other histologic analyses were performed in order to correlate to gastric emptying, cecum arrival, and small intestine transit evaluated by Alternating Current Biosusceptometry. Results showed that prednisone in adult rats increased the frequency of gastric contractions, hastened gastric emptying, slowed small intestinal transit, and reduced mucosal mast cells. Histologically, the treatment with both doses of prednisone decreased villus height, whereas longitudinal and circular muscles and crypt depth were not affected. These findings indicate an impairment of intestinal absorption which may be linked to several GI dysfunctions and symptoms. The relationship between gastrointestinal motor disorders and cellular immunity needs to be clarified in experimental studies since prednisone is one of the most prescribed glucocorticoids worldwide.Our aim was to verify the effects of prednisone related to gastrointestinal motility, intestinal histology, and mucosal mast cells in rats. Two-month-old male Wistar rats were randomly assigned to control group (vehicle) animals receiving saline 0.9% ( Glucocorticoids and their derivatives are the most prescribed synthetic drugs in clinical practice due to their large immunomodulatory activity . Over thFor several gastrointestinal diseases, the role of the mucosal immunity is currently being explored . Due to Adverse effects of prednisone are well-documented for several systems and/or for specific diseases. In mice pulmonary tissue, treatment with prednisone was able to reduce the mucosal mast cell transendothelial migration . HoweverNoninvasive techniques such as Alternating Current Biosusceptometry (ACB) are essential to evaluate GI motor functions, including gastric contractility , gastricMale Wistar rats (250–300 g) were maintained in controlled conditions of temperature (22 ± 3°C), humidity (60 ± 5%), and 12-hour light/dark cycle with access to commercial chow (Purina®) and filtered water ad libitum. All experimental procedures were approved by the Ethics Committee on Animal Research from Federal University of Mato Grosso (protocol number 23108.049862/13-3) and followed the Guidelines for Ethical Conduct in the Care and Use of Experimental Animals.n = 7), and treated group, in which animals received 0.625 mg/kg/day of prednisone (n = 7) or 2.5 mg/kg/day of prednisone (n = 7). Vehicle or prednisone treatments were administered orally during 15 consecutive days, always at the same time period. All analysis were performed at the end of the administration of vehicle or prednisone.Animals were randomly assigned to control group, in which animals received only vehicle (0.9% NaCl)  was used as a nonabsorbable magnetic marker incorporated to laboratory chow. Detailed technical information has been reported earlier [Magnetic monitoring of GI transit and contractility was performed employing the ACB technique  , 14. ACB earlier , 15.After fasting overnight, animals were fed with the magnetically marked chow (1.6 g laboratory chow blended with 0.4 g ferrite powder). ACB sensor was placed on the abdominal surface and the maximum magnetic signal intensity value for both stomach and cecum projection  was recorded. Subsequent measurements were performed in awake rats upon those same points at regular 15-min intervals for at least 5 h .For GI transit measurements, the animals were anesthetized with 75 mg/kg ketamine  plus 2.5 mg/kg acepromazine , intraperitoneally. Animals were then laid in supine and the ACB sensor was placed on the stomach surface towards recording the magnetic signals continuously for 30 minutes at a sampling rate of 20 Hz, by using a multichannel recorder  .After gastric contractility measurements, animals were killed by anesthetic overdose consisting of 240 mg/kg ketamine plus 45 mg/kg xylazine chlorhydrate solution  administered intraperitoneally. Immediately after the anesthetic overdose, the animals were decapitated in order to collect blood and tissues for analysis.3/mm3. Secondly, to differential cell counts, a total of 100 cells were obtained using a blood smear, and the slides were stained with panoptic . Cell populations were differentially counted based on the morphological features and the results were presented in absolute values (103/mm3) [Blood cell count for both control and treated animals groups was carried out by two different methods. Firstly, to analyze the total number of leukocytes, blood samples collected in EDTA-coated tube were diluted 1 : 20 in Turk solution, counted using a Neubauer chamber, and the results were expressed in 10103/mm3) .μm) sections (microtome HM-355S Automatic Microtomes Thermo Scientific) were stained with toluidine blue 0.5% and hematoxylin and eosin (HE). Toluidine blue 0.5% staining sections were used to identify mast cells (MC) in the intestinal mucosa, since MC granules display metachromatic staining after uptake of toluidine blue dye. For each rat, sections of duodenum were randomly selected. Twenty well-oriented villus-crypt units (VCU) were examined per animal and expressed as mucosal MC per VCU [Tissue samples from duodenum were fixed in methacarn , dehydrated in alcohol series, cleared in xylene, and embedded in paraffin. Semiserial 4 micrometers  in which the average amount of magnetic material has been emptied from the stomach, weighted by the area under the emptying curve; the Mean Cecum Arrival Time (MCAT) defined as the time t (min) in which there was an increase in the average amount of magnetic meal that reached the cecum, weighted by the area under the curve between the arrival at the cecum and the cumulative maximum value; and the Mean Small Intestine Transit Time (MSITT) which was determined by the difference between MCAT and MGET [GI transit data was analyzed in Origin® and statistical moments were calculated in order to obtain the Mean Gastric Emptying Time (MGET), as the time and MGET , 20.P) and noise peak intensity (P′) and expressed in decibels (dB) as follows: A = 10 log 10 (P/P′) [Gastric contractility signals were analyzed in Matlab by visual inspection and subsequently, Fast Fourier Transform (FFT) was applied. The highest peak of frequency for each FFT was determined as the gastric dominant frequency and the lowest was the intrinsic noise of the signal. Frequencies were expressed in hertz (Hz) and then converted to cycles per minute (cpm). Amplitude of contraction was determined by the ratio between the intensity of gastric peak (0 (P/P′) .R) was applied to evaluate the relationship between intestinal variables. Only coefficients above 0.80 were considered significant. Differences were considered significant at a p value < 0.05.All results were expressed as mean ± standard deviation (SD) and analyzed by ANOVA, followed by Tukey. Pearson correlation coefficient  group, confirming the immunosuppression in both doses, as expected .Treatment with prednisone, in both doses, has increased (~4.8 cpm) the frequency of gastric contractions compared with the vehicle (4.4 ± 0.4)  whereas  R = 0.8) after prednisone treatment  transducers implanted in laboratory animals and manometric tubes employed in several species, including humans, are often used. Nevertheless, both are invasive approaches, requiring surgery and uncomfortable catheter insertion, respectively . ManometPrednisone administered to rats increased the frequency of gastric contractions, hastened gastric emptying, slowed small intestinal transit, reduced mucosal mast cells, and decreased villus height. These findings indicate an impairment of intestinal absorption which may be linked to several GI dysfunctions and, thereafter, symptoms, which need to be clarified since prednisone is one of the most prescribed glucocorticoids worldwide. Noninvasive techniques such as ACB are promising tools towards evaluating the side effects of time course treatments on GI motility.


Neoandracantha peruensis n. gen. n. sp. are described from the ghost crab Ocypode gaudichaudii collected from the Pacific coast of Peru. While it is uncommon to describe acanthocephalan taxa from immature stages, the presence of clear-cut distinguishing features separating the present material from its nearest congeneric taxa, and the absence of adults, justifies the erection N. peruensis. The new genus is distinguished by having three separate fields of trunk spines. Specimens of N. peruensis have a slender trunk with two anterior swellings, 3 separate fields of spines on the foretrunk swelling, and no genital spines on the hindtrunk. The proboscis of the new species is heavily armored with 21–22 longitudinal rows of 22 hooks each. Hook no. 14 is more robust ventrally than dorsally. Cystacanths of N. peruensis also have a long tubular hindtrunk and the males have diagonal testes in the midtrunk swelling. Specimens of the closely related Andracantha Schmidt, 1975 have anteriorly enlarged pear-shaped Corynosoma-like trunks, only two fields of anterior trunk spines with occasional genital spines, and bilateral or tandem testes. Proboscides of species of Andracantha have considerably fewer hooks that gradually decrease in size posteriorly. The taxonomic component of this work is amplified by metal analysis of hooks and spines that shows a marked amount of magnesium (Mg) in hooks but not in spines. The highest level of sulfur (S) was found in the outer layer of hooks and anterior spines. The metal footprint of hooks and spines varies in different species of acanthocephalans and has an interspecific diagnostic value.The cystacanths of  Corynosoma Lühe, 1904 has members with genital spines in one or both sexes, or only occasionally. As Schmidt [Diplospinifer Fukui 1929 and Southwellina Witenberg 1932.” Schmidt [Andracantha to contain polymorphid species with two fields of trunk spines and genital spines in one or both sexes. The genital spines were noted to occasionally shift anteriorly or be absent. Genital spines were noted by their absence in Andracantha mergi  Schmidt, 1973 and in Andracantha tandemtesticulata Monteiro, Amato, Amato, 2006. Aznar et al. [Corynosoma or Andracantha. We agree. Similarly, we are proposing to establish a new polymorphid genus and species with three fields of trunk spines and uncertain genital spines since all our specimens are immature.The family Polymorphidae Meyer, 1931 includes a wide array of genera that parasitize aquatic birds and mammals. All genera have one thing in common: trunk spines in varied patterns. The confusion surrounding the separation of the various species into recognizable genera based on the trunk spine arrangements was resolved on the basis of the key to the genera in the family developed by Schmidt . Of the  Schmidt  correctl Schmidt  establisr et al.  suggesteNeoandracantha peruensis n. gen., n. sp. collected from the ghost crab Ocypode gaudichaudii Milne-Edwards and Lucas off the Pacific coast of Peru at Callao is its earlier misdiagnosis and reporting once from the same host species in the same location as Andracantha sp. by Vasquez et al. [Profilicollis altmani  Van Cleave, 1947 that infect the sand crab Emerita analoga (Stimpson) also off the Peruvian coast [N. peruensis in local cormorants have not been successful so far. Most species of the related genus Andracantha Schmidt, 1975 have piscivorous birds of the genus Phalacrocorax Brisson as their definitive hosts [The only background history relevant to the discovery of z et al.  in a syman coast , and elsan coast ,10. Atteve hosts . PresentWe examined a total of 1,069 ghost crabs, weighing on average 14 g  and 17 g , and collected in 30 × 30 m grids from various beaches near Lima, Peru. Bióloga Asucena Naupay, Universidad Nacional Mayor de San Marcos, Dr. José Iannacone and his students, Universidad Nacional Federico Villareal, Lima, Peru, and their student assistants and collaborators ,19,25,27Initial collections were made on three beaches of Callao district, located west of the Lima Metropolitan area and bordering Lima Province to the north, east, and south, and the Pacific Ocean to the west. Callao is the same locality from which Vasquez et al.  obtainedOcypode spp. are prevalent); and Playa Gallardo crabs were mostly (81%) juveniles. Overall, we had minimal success and the stochastic environment, climate and other ecological variables were implicated.Descriptions of some of the other collecting sites  follow. Worms were punctured with a fine needle and subsequently stained in Mayer's acid carmine, destained in 4% hydrochloric acid in 70% ethanol, dehydrated in ascending concentrations of ethanol (24 hr each), and cleared in 100% xylene then in 50% Canada balsam and 50% xylene (24 hr each). Whole worms were then mounted in Canada balsam. Measurements are in micrometers, unless otherwise noted; the range is followed by the mean values between parentheses. Width measurements represent maximum width. Trunk length does not include proboscis, neck, or bursa. Line drawing were created by using a Ken-A-Vision micro-projector , which uses cool quartz iodine 150 W illumination. Color-coded objectives, 10 X, 20 X, and 43 X lenses were used. Images of stained whole mounted specimens were projected vertically on 300 series Bristol draft paper , then traced and inked with India ink. Projected images were identical to the actual specimens being projected. The completed line drawings were subsequently scanned at 600 pixels on a USB key and subsequently downloaded to a computer.Type specimens were deposited at the University of Nebraska's State Museum's Harold W. Manter Laboratory (HWML) collection in Lincoln, Nebraska, USA.www.quorumtech.com), establishing an approximate thickness of 20 nm. Samples were placed and observed in an FEI Helios Dual-Beam Nanolab 600 Scanning Electron Microscope  with digital images obtained in the Nanolab software system , and then transferred to a USB key for future reference. Images were taken at various magnifications. Samples were received under low vacuum conditions using 10 KV, spot size 2, 0.7 Torr using a GSE detector.Samples of parasites that had been fixed and stored in 70% ethanol were processed following standard methods . These iStandard methods were used for preparation, similar to the SEM procedure. Specimens were examined and positioned with the above SEM instrument, which was equipped with a Phoenix energy-dispersive X-ray analyzer . X-ray spot analysis and live scan analysis were performed at 16 KV with a spot size of 5 and results were recorded on charts and stored with digital imaging software attached to a computer. The TEAM * software system  was used. Data were stored on a USB key for future analysis. The data included weight percent and atom percent of the detected elements following correction factors.Typical hooks from the proboscis were cut in cross-sections at three levels: tip, middle, and base with a gallium beam . Other hooks were cut along the mid-longitudinal plane with the gallium beam and each cut was scanned twice with X-ray at two positions for the hook (edge and middle). The trunk has three spiny fields: anterior, middle, and posterior. Spines from each area were cut with the gallium beam along the cross-section and then scanned with X-ray for chemical elements. Data were stored on a USB key for future use.A dual-beam SEM with a gallium (Ga) ion source (GIS) was used for the LIMS part of the process. The proboscis hooks were sectioned using a probe current between 0.2 nA and 2.1 nA according to the rate at which the area was cut. The time of cutting was based on the nature and sensitivity of the tissue. Following the initial cut, the sample also went through a milling process to obtain a smooth surface. The cut was then analyzed for chemical ions with an electron beam (Tungsten) to obtain an X-ray spectrum. Results were stored with the attached imaging software then transferred to a USB key for future use. The intensity of the GIS was variable due to the nature of the material being cut.Ocypode Weber, inhabit the sandy shores of tropical and subtropical regions throughout the world. They are mostly nocturnal, inhabiting the deep burrows in the intertidal zone and are generalist scanvengers and predators of small animals. The ghost crab O. gaudichaudii is found along the Pacific coast of the Americas from Guatemala to Chile [The following description is based on the study of 12 cystacanths obtained from 6 out of 1,069 examined ghost crabs (0.56%) from the Pacific coast near Lima, Peru with a maximum of 4 worms per crab. Ghost crabs, genus to Chile ,28.urn:lsid:zoobank.org:act:ACBEF202-8280-4E89-A03F-7B76C0BEAFA8Diagnosis. Polymorphidae. Trunk in 3 segments, foretrunk, midtrunk, and hindtrunk; the first two separated by constriction. Foretrunk slender with 3 fields of spines and prominent middle swelling and including proboscis receptacle and lemnisci. Midtrunk bulbous with no spines and including testes in males and embryonic eggs at its posterior end in females. Hind trunk tubular including distal underdeveloped reproductive system, genital ligaments, and genital terminalia of both sexes. Proboscis with many longitudinal rows of many rooted hooks and unrooted spines; swollen near middle at level of transition between hooks and spines. Proboscis receptacle double-walled inserted at base of proboscis with cephalic ganglion near its middle. Testes diagonal. Cement glands barely discernable. Gonopores terminal in males and subterminal in females. Parasites of crabs off Peruvian Pacific.Neoandracantha peruensis n. sp.Type species: Neoandracantha n. gen. characteristically have 3 fields of spines separated by bare zones in the slender foretrunk which has a middle swelling bearing the middle field of spines. The 3 fields of spines alone set the new genus apart from Andracantha which has only 2 fields of spines on the anterior trunk. The foretrunk and the bulbous spineless midtrunk of the new genus are separated by a constriction and the testes are diagonal compared to being in tandem or bilateral as they are in all 7 species of Andracantha.Cystacanths of the genus urn:lsid:zoobank.org:act:A8A22A5A-05CC-40E3-BBDC-63164301F19BFamily: Polymorphidae Meyer, 1931Neoandracantha n. gen.Genus: Ocypode gaudichaudii Milne-Edwards and LucasType host of cystacanths: ′36″S 77°1′42″W).Type locality: The Pacific Ocean off the Peruvian coast at Lima  collection in Lincoln, Nebraska, Collection No. 139135  and No. 139136 .Andracantha. The specific name describes the geographical location.Etymology: The name of the new genus addresses the relation to the genus Neoandracantha (Polymorphidae). Structures usually relatively larger in females than in males. Trunk in 3 segments; anterior 2 segments (foretrunk and midtrunk) separated by constriction. Foretrunk with middle swelling enclosing proboscis receptacle and lemnisci and bearing 3 fields of spines separated by bare zones : Trunk in 3 regions measuring 13.95–17.30 (15.49) mm in total length. Foretrunk 2. 02–2.92 (2.36) mm long by 0.63–1.15 (0.86) mm wide at middle swelling. Midtrunk 2.12–2.77 (2.38) mm long by 1.00–1.60 (1.21) mm at swelling. Tubular hind trunk 9.50–12.00 (10.75) mm long by 0.35–0.50 (0.42) mm wide at posterior end. See Female (based on 5 whole mounts and 1 specimen used for SEM generation): Trunk in 3 regions measuring 14.50–18.60 (16.27) mm in total length. Foretrunk 2.72–2.87 (2.79) mm long by 0.93–1.17 (1.01) mm wide at middle swelling. Midtrunk 2.12–2.30 (2.23) mm long by 1.00–1.50 (1.19) mm wide at swelling. Tubular hind trunk 10.16–13.00 (11.25) mm long by 0.41–0.62 (0.50) mm wide at posterior end. See Neoandracantha peruensis n. gen., n. sp. is primarily distinguished from species of the closely related Andracantha Schmidt, 1975 by having a slender trunk with two anterior swellings separated with a constriction, 3 prominent fields of spines on the foretrunk swelling separated by aspinose zones, and no genital spines. Adults and cystacanths of most species of Andracantha have anteriorly enlarged pear-shaped Corynosoma-like trunks, only two fields of anterior trunk spines, and occasional genital spines. In addition, cystacanths of N. peruensis have a long tubular posterior trunk and males have diagonally positioned testes in the midtrunk swelling compared to either bilateral or tandem testes in species of Andracantha. Andracantha tandemtesticulata described from the Neotropical cormorant, Phalacrocorax brasilianus (Gmelin) in Southern Brazil [Andracantha that is close to N. peruensis in trunk shape and organization. Nevertheless, it has two fields of spines in the anterior trunk, tandem testes and different proboscis armature.n Brazil  is the oCancer irroratus Say infected with cystacanths of Profilicollis major Lundström, 1942 from 20 and 51 examined crabs; prevalence of 20% and 37%, respectively. Their subsequent examination of 700 and 350 rock crabs from the New Jersey and Delaware coasts over a period of 4 years yielded no parasites. For a better understanding of the distribution and habitats of populations of O. gaudichaudii, see Quijón et al. [The discrepancy between our limited parasite recovery success compared to that of Vasquez et al.  who obtan et al.  and Moscn et al. .Egretta thula (Molina), Guanay cormorant, Leucocarbo bougainvillii (Lesson), royal tern, Thalasseus maximus (Boddaert), American oystercatcher, Haematopus palliates Temminck, and Franklin's gull Leucophaeus pipixcan (Wagler). We, however, believe that adults of Neoandracantha peruensis are similar to the described cystacanths based on corroborating reports. For example, Nickol et al. [Arhythmorhynchus frassoni  Lühe, 1911 from fiddler crabs, Uca rapax, in Florida similar to our cystacanths of N. peruensis. Their specimens had an anterior spined foretrunk “ending in contriction followed by unspined bulbous swelling …. followed by long threadlike hindtrunk”  Witenberg, 1932 with similar morphology to cystacanths of N. peruensis including “two trunk regions separated by a constriction with spiny anterior” from the hairy shore crab Hemigrapsus oregonensis (Dana) from British Columbia, Canada [Bucephalus clangula (L.).” Other polymorphid cystacanths and adults with similar proboscis armature include Corynosoma stanleyi Smales, 1986 which was reported from 3 species of Australian shore crabs , and from one species of mammal, the water rat Hydromys chrysogaster Geoffroy, respectively [Profilicollis novaezalnandensis Brockerhoff and Smales, 2002 from the intertidal crab Hemigrapsus crenulatus (Milne Edwards) and adults from the oystercatcher Haematopus ostralegusfinschi Martins in New Zealand.Comparable findings were found in male and female cystacanths of , Canada . Ching [, Canada  also repectively . Similarectively  demonstrMacracanthorhynchus ingens  Meyer, 1932 and Oligacanthorhynchus tortuosa  Schmidt, 1972. Hook size and proboscis length appear to remain stable through development from cystacanth to adult” [Mediorhynchus grandis Van Cleave, 1916 are fixed by the time worms became infective cystacanths, and Amin [Acanthocephalus dirus Van Cleave (1931), Van Cleave and Townsend, 1936.In non-polymorphid acanthocephalans, “No significant differences were detected in proboscis length and hook length Leidy, 1850) Schmidt, 1972 between cystacanths and adults of “o adult” . Moore [o adult”  asserted850 SchmiN. peruensis n. gen., n. sp. Other species of Acanthocephala have also been described from cystacanths collected from intermediate or paratenic hosts. For example, Corynosoma beaglense Laskowski, Jeżewski, Zdzitowiecki, 2008 was described from the cystacanth stage infecting the body cavity of Champsocephalus esox Günther (Perciformes) in the Beagle Channel [Nybelinia surmenicola Okada in Dollfus, 1929 (Tentacularidae: Trypanorhynchidae) was made from plerocercoids collected from the squid Todarodes pacificus, Steenstrup [While it is uncommon to describe acanthocephalan taxa from immature stages, the presence of clear-cut distinguishing features, especially trunk spination and proboscis armature, separating the present material from its nearest congeneric taxa, in the absence of adults, justifies the erection of  Channel ; the defeenstrup . Thirty-eenstrup . Many moEchinorhynchus baeri Kostylew, 1928 [Rhadinorhynchus oligospinosus Amin and Heckmann, 2017 [N. peruensis, the magnesium (Mg) appeared in marked amounts. Common elements (C and O) were recorded with Mg, P, S, and Ca appearing in the hooks. The highest level of sulfur (S) was found in the outer layer of hooks (edge layer) especially at the middle of the hook. These elements are critical for the mineralization of the hook which creates the hardened outer layer, an apatite, similar to tooth enamel for mammals. Mg probably plays a role in the mineralization of hooks similar to the disulfide bonds formed by S in the protein apatite.The metal profile of hooks and spines in species of Acanthocephala has a taxonomic implication as it will vary by species and can be used as an additional interspecific diagnostic tool. Results of the analysis of mineral content in proboscis hooks are found in nn, 2017 , among oThe data for the cut trunk spines using X-ray analysis (EDAX) can be found in Corynosoma Lühe, 1904 has one field of trunk spines and possibly genital spines in one or both sexes, or only occasionally. Schmidt [Andracantha to contain polymorphid species with two fields of trunk spines and genital spines in one or both sexes, if at all. Aznar et al. [Corynosoma or Andracantha. Our present contribution expands the concepts of polymorphids with one field of spines in Corynosoma to polymorphids with two fields of spines in Andracantha to polymorphids with three fields of spines in Neoandracantha with uncertain genital spines, since all our specimens are immatures. Structures other than trunk spines such as proboscis armature and placement of testes also contribute to the distinction of Neoandracantha n. genus from other polymorphids. We have also shown that size, number and distribution of trunk and proboscis armature in the cystacanths will match those in the adults should any be successfully recovered from their potential bird definitive hosts at a future date. We will continue our efforts to obtain adults from crab-eating birds in the same general areas where cystacanths were collected.Polymorphid genera have trunk spines in varied patterns and genera are recognized based on the trunk spine arrangements . Of the  Schmidt  establisr et al.  suggesteAcanthocephalus lucii . In our other studies [For the first time, we observed recordable amounts of magnesium in the proboscis hooks. Brazova et al.  had simi studies ,12,32, iThe authors declare that they have no conflict of interest.