To evaluate the outcome and complications of transscleral suture-fixated posterior chamber intraocular lens (PCIOL) implantation combined with Ahmed glaucoma valve (AGV) surgery in Asian eyes.  This was a retrospective study that included 22 eyes from 22 participants. The surgeries were performed at Singapore's National University Hospital. Participants underwent an Ahmed tube surgery, together with transscleral suture-fixated posterior chamber intraocular lens.  Complete success was defined as 6 ≤ intraocular pressure (IOP) ≤ 21 mmHg without medications at the last follow-up visit, with no reoperation required and no progression to no perception of light (NPL). p < 0.001) and mean number of intraocular pressure-lowering medications . There was no significant change in visual acuity [1.43 ± 1.21 (LogMAR) versus 1.09 ± 1.31 (p = 0.204)]. Sixteen eyes (72.7%) achieved complete success. The 3 commonest complications were bullous keratopathy, choroidal detachment, and displacement of intraocular lens.  At the last follow-up, there was a significant reduction in mean IOP (22.4 ± 6.5 mmHg versus 13.9 ± 3.9 mmHg;  This technique showed good success for intraocular pressure control and vision preservation. Postoperative complications were relatively common although most were self-limiting. Patients at increased risk of trabeculectomy failure may be suitable for this procedure. Compared to anterior chamber lens, posterior chamber intraocular lens (IOL) implantation has several advantages including lower risk of corneal endothelial damage, superior visual outcomes, and less damage to anterior chamber angle structures , 2. The Previous studies explored the option of transscleral suture-fixated PCIOL implantation combined with a trabeculectomy and found that this technique resulted in good intraocular pressure (IOP) control as well as improvement in visual acuity , 9. To op < 0.006), but there was no significant difference in IOP reduction between the treatment groups after 3 months [In the tube versus trabeculectomy (TVT) study, Gedde  compared3 months . Additio3 months .The TVT study highlights that tube shunts have the potential to play an increasing role in the surgical management of glaucoma . MoreoveThe retrospective study consisted of 22 consecutive eyes of 22 glaucoma patients who underwent transscleral suture-fixated PCIOL implantation in combination with AGV surgery and had a minimum of 3 months of follow-up. The surgeries were performed by 4 consultant surgeons between 2006 and 2015 at National University Hospital, Singapore. The study adhered to the principles laid out in the Declaration of Helsinki and obtained ethical approval from the National Healthcare Group Domain Specific Review Board (NHG-DSRB).Among our study patients, the indication for surgery was the presence of both glaucoma and inadequate posterior capsule support, as shown in The data recorded included gender, race, age at surgery, systemic conditions, type of cataracts, type of glaucoma, slit lamp biomicroscopic evaluation, best-corrected visual acuity (BCVA) using Snellen chart, IOP using Goldmann applanation tonometry, number of IOP-lowering medications, intraoperative details, postoperative complications, and reoperation.Complete success was defined as 6 ≤ IOP ≤ 21 mmHg without IOP-lowering medications at the last follow-up visit, with no reoperation required and no progression to NPL. Failure was defined as IOP > 21 mmHg or <6 mmHg, or if reoperation was required, or if there were IOP-lowering medications at the last follow-up visit, or if there was progression to NPL .All patients underwent an AGV insertion, together with transscleral suture-fixated PCIOL (either via anterior segment or posterior vitrectomy). All surgeries were performed at the National University Hospital, Singapore.Conjunctival peritomyAhmed tube patency verifiedPlate of seton placed in subtenon space between recti muscles and secured with nylon suturesTrack of tube into anterior chamber created with 23-gauge needleEnd of tube trimmed to desired length and inserted into the anterior chamberTube secured with nylon sutures to the underlying scleraTutopatch  glued over path of tube and secured with Tisseel glue Conjunctiva closure with nylon suturesBriefly, for AGV surgery, the surgical steps includedIntracapsular cataract extractionAnterior vitrectomy performedThree-piece IOL sutured to sclera with 10/0 proleneTransscleral suture-fixated PCIOL implantation (anterior segment)Trans-pars plana vitrectomy with lensectomy performedPupil constricted with Miostat and IOL inserted from clear corneal wound10/0 prolene sutures applied to fixate three-piece IOL to scleraTransscleral suture-fixated PCIOL implantation (posterior vitrectomy)Upon discharge, patients were prescribed with antibiotic and steroid eye drops, to be used three-hourly to four times daily for the first postoperative week. Antibiotics were stopped one month after the surgery and the steroids were continued with slow taper. In general, all patients were reviewed at approximately one day, one week, one month, three months, and every six months postoperatively.p value < 0.05 was taken to be statistically significant.Statistical analysis was performed using SPSS version 20 . Patient demographics and baseline characteristics were analysed using descriptive statistics; the Wilcoxon signed-rank test was used to compare preoperative and the last visit measurements of IOP, number of IOP-lowering medications, and visual acuity. Visual acuity measured using the Snellen chart was converted to logarithm of minimum angle of resolution (LogMAR) for ease of statistical analysis. A Kaplan-Meier curve was used to assess the probability of success over time. A The eyes were followed up for a mean duration of 29.0 ± 19.4 months (median = 27 months), with a range of 3 to 66 months. The overall follow-up rates were 90.6% at 6 months, 77.3% at 12 months, 68.2% at 18 months, and 54.5% at 2 years. Five patients (22.7%) missed one or more follow-up visits, but were not entirely lost to follow-up—they were not excluded from the study. The right eye was operated on in 8 patients (36.4%) and the left eye in 14 patients (63.6%). Nine of the patients were diabetic (40.9%) and 12 of the patients were hypertensives (54.5%). There was a statistically significant reduction in mean IOP from preoperation to the last follow-up. At the last follow-up, 21 eyes (95.5%) had an IOP ≤ 21 mmHg. Only 1 eye had a pressure of >21 mmHg (22 mmHg). This patient experienced a clot over tube causing tube blockage and vitreous hemorrhage immediately postoperatively, but this was self-limiting without further intervention.p = 0.204). At the last follow-up, 3 eyes had a BCVA 6/6 (13.6%). One eye had NPL at the last visit, but preoperatively could only perceive light (PL). It did not experience any complications nor require any further surgical intervention. Six eyes were found to have poorer vision at the last follow-up than preoperatively, but only 3 eyes lost more than 2 lines of vision. Of these 3 eyes, 1 eye experienced retinal detachment, 1 eye experienced tube exposure and endophthalmitis, and 1 eye experienced choroidal detachment, cystoid macular edema, and corneal decompensation.The BCVA of the eyes showed a statistically insignificant improvement, from 1.43 ± 1.21  preoperatively to 1.09 ± 1.31  at the last visit (p < 0.001). Only 1 patient was still taking a single IOP-lowering medication at the last visit. This patient experienced posterior dislocation of the IOL 1 week after the operation and underwent left trans-pars plana vitrectomy, PCIOL explantation, and anterior chamber intraocular lens (ACIOL) implantation 1 week later.The mean preoperative number of IOP-lowering medications showed a statistically significant decrease, from 2.45 ± 1.30  to 0.05 ± 0.21  at the last follow-up  at the last follow-up. Treatment failure in our study population was due to requirement of reoperation (4 eyes), IOP > 21 mmHg at the last visit (1 eye), IOP < 6 mmHg at the last visit (1 eye), BCVA NPL at the last visit (1 eye), and taking IOP-lowering medication at the last visit (1 eye). This is the first study evaluating the outcomes and complications of transscleral suture-fixated PCIOL combined with Ahmed tube implantation. More than 70% of the patients in our study had secondary glaucoma from previous ocular trauma, surgery, uveitis, and neovascular glaucoma, all of which placed them at increased risk of trabeculectomy failure. We will discuss our findings in relation to the studies of Shin et al. and David et al., all 3 of which are summarized in Among our study participants, the success rate was 72.7% (16 eyes). The most common reason for failure was requirement of reoperation. Shin et al. had 3 different criteria for success. Their least stringent criteria were as follows: stable visual acuity (improved or within two Snellen lines of preoperative BCVA) and a postoperative IOP of 21 mmHg or less, but with or without the use of medications, 5-fluorouracil (5-FU) needling revision, or incisional glaucoma surgery. Their intermediate criteria included all of the above except that it did not allow for incisional glaucoma surgery, and their most stringent criteria, in addition, did not allow for 5-FU needling revision. Overall, depending on which criteria was applied, 46% to 68% of the patients had both stable visual acuity and satisfactory pressure control at the last postoperative visit [Our study suggested that our success rates are superior to the rates seen in their study with lower risk of reoperation required. However, this may also have been due to our shorter mean follow-up time (29.0 months versus 38.5 months). Another study done by David et al. evaluated the outcomes of transscleral suture-fixated PCIOL combined with trabeculectomy in patients with subluxated/dislocated lens. In their study, complete success was achieved in 80% of the participants at 2 years follow-up. This is similar to ours, but it must also be noted that their criteria for complete success were more lenient than ours—they allowed for IOP < 6 mmHg, reoperation, as well as progression of vision to NPL.Among our study participants, transscleral suture-fixated PCIOL combined with Ahmed tube implantation was effective in reducing IOP and reducing number of IOP-lowering medications. This is consistent with the findings of Shin et al. and David et al. In terms of vision, the BCVA of our study's eyes showed that visual acuity did not change significantly after surgery. On the other hand, Shin et al. and David et al. found a statistically significant improvement in BCVA. Although our improvement was statistically insignificant, it could be attributed to the small sample size . It is also possible that the lack of a significant improvement in vision is due to the fact that the primary indication for surgery in our patients was raised intraocular pressure and not poor preoperative vision.In terms of the complication profile, our study had 36.4% of patients experienced postoperative complications. In contrast, Shin et al. found that 14% of the eyes experienced postoperative complications, whereas David et al. found that 13.7% of the eyes experienced early postoperative complications and 4% of the eyes experienced late postoperative complications. However, it is worthy to note that most of our complications were self-limiting did not require reoperation and did not adversely affect vision preservation or IOP control in the long run. The complications present in our study were similar to those found by Shin et al. and David et al., such as choroidal detachment, retinal detachment, PCIOL dislocation, and hypotony. Shin et al. found 2 patients with suprachoroidal hemorrhage, which was a complication that did not occur in our study population, but associated with trabeculectomy surgery.The major limiting factor of our study was its retrospective nature. Therefore, we could not standardize the method and contents of documentation and this gave rise to missing information for some of the study participants. This inconsistency in documentation may also have been due to the fact that the operations were not all performed by a single ophthalmologist. Secondly, due to our small sample size, we could not perform subgroup analysis. Moreover, some statistically insignificant results could possibly become statistically significant with a larger sample size. Finally, because our study comprised almost exclusively of Ahmed tube implantation, the findings may not be applicable to other tubes such as the Baerveldt tube.In conclusion, we found that transscleral suture-fixated PCIOL combined with an AGV surgery showed good success rates for IOP control and vision preservation. Although postoperative complications were relatively common, most were self-limiting and did not require reoperation or adversely affect vision preservation or IOP control in the long run. Further studies in other populations that evaluate the outcome of transscleral suture-fixated PCIOL with tube surgery would be helpful, especially if they have a larger sample size. In particular, subgroup analysis would also be important to investigate the predictors of complications and reoperation, which are critical to identifying the group of patients who are most likely to benefit from this procedure.


Zinc (Zn) and copper (Cu) may play a pivotal role in the pathogenesis of diabetes and diabetic complications by mediating oxidative stress. Both Zn deficiency and excess of Cu are associated with an increased risk of type 2 diabetes and cardiovascular disease. We aimed to investigate the relationships between serum Zn/Cu ratio and glycemic status, renal function, and metabolic parameters in patients with and without type 2 diabetes.We conducted a cross-sectional study on 355 subjects (149 type 2 diabetic and 206 non-diabetic) in whom serum Zn and Cu levels were measured at the same time. Associations between serum Zn/Cu ratio and clinical data were evaluated using multiple regression analysis. We also evaluated associations between serum Zn/Cu ratio and the prevalence of type 2 diabetes and glycemic control by multivariate logistic regression analysis.p = 0.014). Plasma B-type natriuretic peptide levels were negatively associated with serum Zn/Cu ratio after adjustment for age, sex, and BMI . In patients with type 2 diabetes, serum Zn/Cu ratio was negatively associated with plasma HbA1c levels after adjustment for age, sex, and BMI . In addition, multivariate logistic regression analysis revealed that the highest quartile of serum Zn/Cu ratio was associated with a reduced risk of poor (HbA1c ≥ 7%) glycemic control  in patients with type 2 diabetes.Serum Zn/Cu ratio was positively associated with estimated glomerular filtration rate after adjustment for body mass index (BMI) (β = 0.137, Serum Zn/Cu ratio was favorably associated with renal function in all subjects and glycemic control in patients with type 2 diabetes. The Zn/Cu ratio, in addition to the individual serum levels of trace elements, is important for metabolism in humans. Diabetes is a metabolic disease characterized by defects in insulin secretion, insulin sensitivity, or both. Trace elements, such as zinc (Zn) and copper (Cu), may play a pivotal role in the pathogenesis of diabetes and diabetic vascular complications by mediating oxidative stress –4. SeverSLC30A8), regulates insulin secretion  who measured both serum Zn and Cu at our hospital were investigated retrospectively. Exclusion criteria were type 1 diabetes, malnutrition [serum albumin (Alb) < 3.0 g/dl], and anemia [plasma hemoglobin (Hb) levels < 10 g/dl]. Chronic kidney disease was defined as an estimated glomerular filtration rate (eGFR) less than 60 mL/min/1.73 mdiabetes . Brieflydiabetes .The patients were anonymized to protect their personal information. The study protocol was approved by the Medical Ethics Committee of the National Center for Global Health and Medicine Kohnodai Hospital (Reference No. NCGM-G-001912), and the study was performed in accordance with the Declaration of Helsinki.Height and weight were measured using a rigid stadiometer and calibrated scales . Body mass index (BMI) was calculated as body weight in kilograms divided by the square of body height in meters.®, ABBOTT JAPAN Co., Ltd., Tokyo, Japan).We measured blood cell count, Alb, blood urea nitrogen, creatinine, eGFR, Zn, and Cu. Serum Zn and Cu levels were measured by atomic absorption spectrophotometry  , 24. eGFBody composition was analyzed using a bioelectrical impedance analysis device . This method is based on the principle that lean body mass contains higher water and electrolyte content than fat tissue; hence, these tissues can be distinguished by electrical impedance. Segmental body composition was estimated using a patented 8-point tactile electrode system. The device uses six frequencies  and produces 30 impedance values for five body segments: the right and left upper extremities, trunk, and right and left lower extremities . A previt-test. Furthermore, multivariate logistic regression analyses were performed to analyze the association between the quartile of serum Zn/Cu ratio and glycemic control  in patients with type 2 diabetes and to determine odds ratios and 95% confidence intervals (CI). p values < 0.05 were considered statistically significant.Statistical analyses were performed using SPSS version 23 . All values are expressed as mean ± SD. Pearson’s correlation coefficient was calculated to analyze the association of serum Zn/Cu ratio with physical, biochemical, and physiological data. Multiple regression analysis was performed to test independent correlations between serum Zn/Cu ratio and clinical data. Differences in clinical data between patients with and without type 2 diabetes were analyzed using the paired p < 0.001), hypertension (p < 0.001), and CVD (p < 0.001) was higher and the prevalence of endocrine disorders (p = 0.005) and autoimmune diseases (p < 0.001) was lower in patients with type 2 diabetes than in those without (by chi-square test).A total of 355 subjects (151 men and 204 women) were included in this study. The mean age was 61.1 ± 17.6 years. The number of patients with type 2 diabetes was 149 (42.0%). The number of patients with dyslipidemia, hypertension, CKD, and liver diseases such as fatty liver were 117 (33.0%), 94 (26.5%), 87 (24.5%), and 35 (9.9%), respectively. Seventy two patients (20.3%) suffered from autoimmune diseases such as Hashimoto disease, Sjögren’s Syndrome, and rheumatoid arthritis. Thirty-three patients (9.3%) suffered from endocrine disorders such as adrenal deficiency and hyper/hypothyroidism. Eighteen patients (5.1%) had malignant diseases, such as lung cancer, prostate cancer, breast cancer, gastric cancer, and colon cancer. Eleven patients (3.1%) had neurodegenerative diseases, such as Parkinson disease and Alzheimer dementia. The number of patients who had a history of CVD was 29 (8.2%). Patient characteristics are summarized in Table Serum Zn/Cu ratio was inversely correlated with age and plasma BNP levels, whereas it was positively correlated with height, weight, serum Alb levels, and eGFR. Serum Zn/Cu ratio was also positively correlated with red blood cell count, Hb, Ht, and skeletal muscle mass Table .p < 0.001 and β = 0.237, p = 0.002, respectively). Plasma BNP levels were also negatively associated with serum Zn/Cu ratio . In addition, eGFR was positively associated with serum Zn/Cu ratio after adjustment for BMI . However, independent associations between serum Zn/Cu ratio and other biochemical parameters were not detected.Positive associations of serum Zn/Cu ratio with serum Alb levels and Hb remained after adjustment for age, sex, and BMI . However, there were no differences in the prevalence of other diseases, such as hypertension (p = 0.065) and CKD (p = 0.23), between patients with and without type 2 diabetes. In patients with type 2 diabetes, serum Zn/Cu ratio was positively correlated with height , serum levels of Alb , Hb , and Ht  and negatively correlated with age , plasma HbA1c levels  . Positive associations of serum Zn/Cu ratio with serum Alb levels and Hb were observed after adjustment for age, sex, and BMI , and serum Zn/Cu ratio remained negatively associated with plasma HbA1c levels after the same adjustments . However, significant associations between serum Zn/Cu ratio and other biochemical data disappeared following statistical adjustments.The prevalence of dyslipidemia was higher in patients with type 2 diabetes than that in patients without diabetes p < 0.00. Howeverp = 0.025) in patients with type 2 diabetes  for investigating the association between serum Zn/Cu and insulin and confounding effects of oral hypoglycemic agents and diet. We are unable to confidently explain the negative association observed between serum Zn/Cu ratio and BNP in this study. Thus, further studies are required to elucidate the mechanisms underlying the association between serum Zn/Cu ratio and BNP.The negative association observed between serum Zn/Cu ratio and plasma BNP levels in this study may be mediated by insulin sensitivity. An inverse association between natriuretic peptides and insulin resistance has been reported by previous studies –44. BNP ynthesis  and the ynthesis . Recentlynthesis . The invSeveral studies have demonstrated decreased serum/plasma Zn levels in patients with type 2 diabetes compared with healthy individuals –55; on tLow HDL-C levels in patients with type 2 diabetes may have been observed as serum HDL-C levels have been shown to decrease with impaired function in type 2 diabetes . Patientp = 0.436), whereas serum Cu levels were associated with glycemic control . However, the high serum Zn/Cu ratio was associated with good glycemic control in patients with type 2 diabetes. This result suggests that not only Zn and Cu status alone but also Zn/Cu ratio should be considered when evaluating the relationship between these trace elements in patients with diabetes.The observed associations of serum Zn/Cu ratio with lower HbA1c levels and reduced risk of poor glycemic control (HbA1c ≥ 7.0%) in a population of patients with type 2 diabetes are noteworthy findings of this study. Although excess of intracellular Zn can trigger oxidative stress from mitochondria and lead to neuronal degeneration , Zn has This study had several limitations. First, a causal relationship between serum Zn/Cu ratio and renal function and/or glycemic control could not be evaluated because of the retrospective and observational nature of this study. Second, we were unable to evaluate the utility of other biomarkers in assessing Zn and Cu status, such as urinary and other tissue concentrations of Zn and Cu. Zn and Cu concentrations are known to be affected by other factors, such as inflammation, and fasting or postprandial states , 61. HowThe findings of this study demonstrate that serum Zn/Cu ratio is significantly associated with renal function in all subjects and glycemic control in patients with type 2 diabetes. Serum Zn/Cu ratio was negatively associated with plasma BNP levels. Imbalance between Zn and Cu levels induces oxidative stress and insulin resistance, which may lead to progression of diabetes and diabetic complications. Zn/Cu ratio, in addition to individual levels of each trace element status alone, appears to have an important effect on metabolism, indicating that these trace elements may play a key role in the pathogenesis of metabolic diseases. We, clinicians should note that serum Zn/Cu ratio could be a better indicator for human metabolism as compared with Zn or Cu status alone.HH performed the study, conducted the data analyses, and drafted and revised the manuscript. YK contributed to the data collection and analyses. HY critically reviewed the manuscript and the scientific interpretations of study results. All the authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study sought to determine the effect of the recombinant human growth hormone (rhGH) treatment of Turner syndrome (TS) on height outcome.We searched in MEDLINE, EMBASE and Cochrane Central Register of Controlled Trials and Cochrane Database of Systematic Reviews. A literature search identified 640 records. After screening and full-text assessment, 11 records were included in the systematic review. Methodological quality was assessed using the Cochrane Risk of Bias tool. RevMan 5.3 software was used for meta-analysis. We also assessed the quality of evidence with the GRADE system.P < 0.001, I2 = 4%; P = 0.18), height standard deviation (HtSDS)  and height velocity (HV) . There was a small increase in bone age  after rhGH therapy for 12 months. What is more, the rhGH/oxandrolone combination therapy suggested greater final height , increase and faster HV , with no significant increase in HtSDS and bone maturation compared with rhGH therapy alone.Compared with controls, rhGH therapy led to increased final height (MD = 7.22 cm, 95% CI 5.27–9.18, For TS patients, rhGH alone or with concomitant use of oxandrolone treatment had advantages on final height. Turner syndrome (TS) affects about one in 1500–2500 live-born females , 7, 8.Recombinant human growth hormone (rhGH) has been shown to increase growth and final height in girls who have Turner syndrome , 10, 11.Treatment with rhGH, alone or in combination with the anabolic steroid oxandrolone, has been recommended for children with TS to improve final height. However, final height benefit remains uncertain. Some studies have been reported with optimistic responses to rhGH treatment alone , 17 or wThe aim of the present study was to evaluate height outcome of rhGH alone or with concomitant use of oxandrolone treatment in patients with TS, by using the meta-analytic approach as previously described.A comprehensive search of several databases from each database’s inception to March 30th, 2017, any language was conducted. The databases included PubMed, MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials (CENTRAL) and Cochrane Database of Systematic Reviews. The search strategies were (growth hormone or somatotropin or somatropin or somatotrophin or somatrophin or Pituitary Growth Hormone or Recombinant Growth Hormones) and (Turner syndrome or Ullrich-Turner Syndrome or Bonnevie Ullrich Syndrome or Turner’s syndrome) and publication type  as limiter. We also searched the reference list of all published original articles and several review articles we found for additional references. Publications arising from the same study group on the same patient cohort were considered as a single study for the purpose of this analysis. The study of this protocol-based review was consistent with the PRISMA statement.Studies were considered eligible if they met the following criteria: (1) randomized controlled trials (RCTs); (2) the participants had to have TS confirmed by karyotype; (3) the active intervention was rhGH: that is, biosynthetic human growth hormone, with a sequence identical to that of human growth hormone, marketed under any brand name; (4) intervention and comparison, rhGH alone vs placebo/no treatment, rhGH plus oxandrolone combination therapy vs rhGH alone therapy. We excluded uncontrolled studies, case series, cross-sectional studies and studies with short follow-up duration of less than 1 year. Only full-text articles were included; restriction was placed on the language of studies published in English.Primary outcomes were final height  and height standard deviation (HtSDS) . Height velocity (HV), defined as increment in height per year in centimeters, and bone age  were secondary outcome measures of this study. Data were abstracted by a single reviewer and checked by a second reviewer.Two investigators (P L and F C) independently reviewed the articles and selected eligible studies according to the inclusion criteria for eligible studies. Irrelevant studies were excluded. Detailed information from each included studies was recorded by two authors independently. The following information was extracted from each study: name of first author, year of publication, country in which the study was conducted, number of participants of treatment and control group, initial height, final height, HtSDS, bone age and height velocity. Data extraction was done by two reviewers (P L and F C) with any disagreements resolved through discussion with a third reviewer (L X).s.d.). When information was reported for more than one subpopulation in one study, each subpopulation was treated as a separate comparison in our meta-analysis. A random-effects model was employed to estimate weighted mean differences for HtSDS due to its different assays, calculated as the mean difference between groups. The heterogeneity of the included studies was evaluated with the Cochran Q and the I2 statistic. For the Q statistic, P < 0.10 was considered statistically significant for heterogeneity. The I2 statistic was assessed using the following ranges as guidelines: no heterogeneity (I2 0–25%); moderate heterogeneity (I2 25–50%); large heterogeneity (I2 50–75%) and extreme heterogeneity (I2 75–100%) When either Q statistics (P < 0.1) or I2 statistic (>50%) indicated heterogeneity between studies, the random-effects model was preferred. Otherwise, the fixed-effect model was recommended. Statistical analyses were performed using RevMan 5.3 software.Data were summarized statistically if they were available, sufficiently similar and of sufficient quality. All data should be expressed as means with standard deviations (Two authors (F C and L X) independently assessed the risk of bias. Risk of bias was assessed by using the Cochrane Collaboration’s tool 15. Each study was assessed and scored as ‘high’, ‘low’ or ‘unclear’ risk of bias to the following criteria: random sequence generation; allocation concealment; blinding of participants and personnel; blinding of outcome assessment; incomplete outcome data; selective reporting and other bias. Blinding of patients and clinicians was extremely difficult and generally not feasible in these trials, and we judged that the primary outcome was less prone to be influenced by lack of blinding. Therefore, studies with high risk of bias for any one or more key domains except blinding were considered as at high risk of bias; while studies with low risk of bias for all key domains except blinding were considered as at low risk of bias; otherwise they were considered as at unclear risk of bias. Detailed method for the assessment of risk of bias is described in Fig. 4.Quality of evidence was assessed per outcome, independently by two individuals (P L and L X), using the GRADE guidelines for rating the quality of evidence , 29, 30.The initial searches identified 640 articles. A total of 586 publications were excluded based on review of the title and abstract for several reasons, including study type , population (children) or short duration of GH treatment (<12 months). Of the remaining 54 potentially relevant publications, 10 studies were non-randomized controlled study, 2 articles were conference abstracts, 31 of them do not meet our inclusion criteria. After all, 11 publications remained that described the results of 9 trials that met the clinical inclusion criteria . Nine trP < 0.001, I2 = 44%; P = 0.18, final height; standardized mean difference (SMD) = 1.22, 95% CI 0.88–1.56, P < 0.001, I2 = 49%; P = 0.14, HtSDS). Similar to HV, no significant difference was seen in initial HV in two studies . Three studies . There was significant heterogeneity, therefore, a random-effects model of analysis was used.We identified five studies , 32, 33  studies ; however studies , 17, 33 P > 0.05). Four studies , although baseline height was similar . In parallel with the increases in final height, rhGH with OX-treated subjects had significantly greater increases in HV than did the rhGH alone group . There was significant heterogeneity; therefore, a random-effects model of analysis was used. Meanwhile, we also observed the effects of rhGH in combination with OX on bone age and HtSDS; however, no significant difference was seen between the two groups after treatment (P > 0.05).As shown in  studies , 34, 35 As shown in We have performed a meta-analysis of clinical studies investigating the effect of rhGH therapy in TS patients. The current study was focused on a stringent evaluation of the efficacy of rhGH alone or with concomitant use of oxandrolone in TS. For this reason was limited to randomized controlled trials.et al. . With respect to rhGH dose, treatment was generally administered at fixed doses, ranging from physiological (0.2 mg/kg/week) to supraphysiological (0.43 mg/kg/week) doses. Similar to rhGH, OX dose ranged from 0.03 to 0.125 mg/kg/day doses. Only small studies compared various rhGH dosages , 39; theA limitation of the present meta-analysis is the relatively small number of available studies and the lack of sufficient data on final height. Subgroup analyses or meta-regression, using study level covariates, could not be performed because of the relatively small number of available investigations. Although a blinded study is less likely to be done for rhGH is approved and standard practice for girls with TS, larger studies of longer duration using a blinded, placebo-controlled design are needed to clarify these issues. Second, the trial reporting final height had lost some participants at the time of reporting. It is possible that treated girls who have not achieved final height would be more likely to be excluded. Similarly, girls in the control group who were growing more slowly might be more likely to leave the trial.In conclusion, the findings of the present meta-analysis suggest that rhGH alone or with the concomitant use of oxandrolone therapy in TS patients may improve final height.The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.This work did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.P L and F C conceived and designed the experiments. P L, L X and H L carried out the experiments and calculations. P L and F C wrote and edited the paper. All authors reviewed the manuscript.


Maternal serum samples were collected and used to measure phthalate metabolites and THs. Covariate-adjusted linear regression analyses showed that natural log (Ln)-transferred concentrations of MBP and LnMEHP were negatively associated with TT4 . Stratification analyses by gestational weeks showed significant associations of LnMBP and LnMEHP with TT4 in gestational weeks 5 to 8, 9 to 12, and 13 to 20. Our findings suggest an association of serum phthalates with lower TT4. The influence of MBP and MEHP on TT4 concentrations throughout the early pregnancy may begin from the embryonic stage .Limited studies have consistently shown an association of phthalates exposure with thyroid hormones (THs) in pregnant women. However, it remains unknown on which specific phthalates can affect THs and whether any effects could differ by gestational age. In the present study, we investigated associations between serum concentrations of phthalate monoesters [monoethyl phthalate (MEP), mono-( Di-(2-ethylhexyl) phthalate (DEHP) is usually used for polyvinyl chloride material, food packaging and medical device3. Because phthalates are not chemically bound to product structures, they release into environment such as air, water, and foods and become widespread contaminants. Human exposure to phthalates is ubiquitous6, and their metabolites have been detected in a wide range of biological samples including urine, blood, placenta, amniotic fluid, and breast milk among humans7.Phthalates are industrial chemicals that are extensively used as plasticizers in a variety of consumer products. For instance, diethyl phthalate (DEP) and di-n-butyl phthalate (DBP) are used for personal care and pharmaceutical products9. An increasing number of studies have indicated that phthalates may disrupt the hormone system10. Limited studies16 have indicated that phthalates may have antagonistic effect on the thyroid function both in vivo and in vitro through a variety of mechanisms, such as down-regulation of the human sodium/iodide symporter (NIS) promoter17. It is well known that thyroid hormones (THs) deficiency during early pregnancy would result in neural deficits22. For instance, either low or high maternal free thyroxine (FT4) concentrations during early pregnancy (<18 weeks’ gestation) impairs IQ and decreases grey matter and cortex volume in children19. de Escobar et al. 200720 reviewed that THs changes in mother during the first half of pregnancy potentially damaged fetal brain development. Therefore, exposure to phthalate may reduce fetal THs availability through maternal THs signaling24.Recently, attentions have been focused on the effects of various health risks in susceptible populations including pregnant women and infants28. Huang et al. 28 found urinary monobutyl phthalate (MBP) exposure was negatively associated with serum total thyroxine (TT4) and FT4 in early pregnancy. Johns et al. 201526 observed an inverse association between DEHP and FT4 during gestational weeks 24 to 28. Similarly, our previous study25 showed negative associations of urinary monobenzyl phthalate (MBzP), mono(2-ethylhexyl) phthalate (MEHP) and mono(2-ethyl-5-hydroxylhexyl) phthalate (MEHHP) with serum TT4 and FT4, whereas positive associations of MEHP and MEHHP with thyroid stimulating hormone (TSH). However, Kuo et al. 201516 observed positive correlations between MBP, MBzP, and mono(2-ethyl-5-oxohexyl) phthalate (MEOHP) and FT4 concentrations in the first trimester of gestation. A repeated measures analysis 27 revealed that MEHP and mono-3-carboxypropyl phthalate (MCPP) were positively related to TT4 and FT4, respectively, whereas MEHP, mono-iso-butyl phthalate (MiBP) and MCPP were negatively associated to TSH. Therefore, these findings suggested that the direction of the associations of THs phthalates with THs phthalates varied during pregnancy27. Which kind of phthalates and when phthalates affect THs are still inconclusive.To date, a few studies have reported the potential association of phthalate exposure with thyroid function in pregnant womenOn the basis of the China-Anhui Birth Cohort (C-ABC), the objective of this pilot study was to investigate the cross-sectional association between prenatal serum concentrations of phthalate monoesters and maternal THs concentrations.2. Most women lived in urban areas (79.0%). Only 27.9% of women were none-passive smokers  years and 12.0  ±  3.9 (range: 5−20) weeks, respectively. The pre-pregnancy body mass index (BMI) was 20.1  ±  2.3 kg/mPhthalate metabolites and THs concentrations were measured in the serum samples Table . All theβ  =  0.010, p  =  0.020 and β  =  0.012, p  =  0.031, respectively) after adjusting for potential confounders. LnMBP, LnMEHP, and LnPAE were negatively associated with TT4 .Linear regression analyses Table  showed tβ  =  0.019, p  =  0.049), and an inverse association of LnMBP, LnMEHP, and LnPAE with TT4  were observed as early as gestational weeks 5 to 8 and followed by gestational weeks 9 to 12 and 13 to 20. No significant association of any phthalate metabolites with TSH was observed.The addition of a product interaction term of gestational age with each phthalate metabolite to the model indicated that the associations of phthalate metabolites with TSH, FT4, or TT4 did not differ by time stages assessed in this study (data no shown). Stratification analyses 31 and the differences in laws and regulations against application of phthalates in different countries.In the present study, the detection rates of the three phthalate metabolites in serum were 86%−100%. The medians of serum MEP, MBP, and MEHP were 0.2, 5.97, and 6.14 ng/mL, respectively. The result suggested that Chinese women during gestational weeks 5 to 20 were widely exposed to low concentration of DEP, DBP, and DEHP. It was expected that concentrations of phthalate metabolites in urine were higher than those in serum. However, our reported concentrations of serum phthalates were different from those in other studies. The geometric means of serum MEP, MBP, and MEHP were 67.8, 14.7, and 1.39 ng/mL respectively, in females from the 2009–2010 National Health and Nutrition Examination Survey4 concentrations34. A few epidemiological studies explored the associations of prenatal phthalates exposure with maternal thyroid function, which was assessed by metabolites concentrations in urine samples28. Three studies reported that maternal phthalates exposures  were associated with reduced concentrations of TT428, which were consistent with our results. However, Johns et al. 201627 reported that increased MEHP was associated with an increase of TT4. No significant associations of maternal TT4 concentrations were found by Kuo et al. 201516 and John et al. 201526. Three previous studies revealed that some phthalates  exposure were related to decreased FT4 concentrations26, whereas two investigations reported opposite results27. No significant relationships were observed for TSH in most previous studies. Our previous study25 found that MEHP and MEHHP were positively associated with TSH, while Johns et al. 201627 observed an inverse association of MEHP, MiBP, and MCPP with TSH. Obviously diverse relationships between phthalates and THs were showed. We speculated that there were several potential reasons for these inconsistent findings. First, these studies were based on different sample sizes. Only our previous study recruited over 500 women 25. Second, urine samples were collected in different time/gestational weeks. Two studies25 obtained urine samples in the first trimester of pregnancy, and three studies28 in the second trimester of pregnancy. Johns et al. 201627 averagely collected urine in 10th, 18th, 26th and 35th weeks’ of gestation for a repeated measures analysis.Our results showed negative associations of maternal serum LnMBP and LnMEHP with TT4 concentrations, which occurred as early as gestational weeks 5 to 8. Similarly, the association of “LnPAE” with TT4, which combined the three metabolites into a single class of chemicals, was also identified. Positive associations of serum LnMBP and LnMEHP with LnFT4 were also found. Data on the association of serum phthalates with thyroid function in pregnant women are limited. Previous animal studies showed that rats fed DEHP through the diet displayed histopathologic alterations in the thyroid indicative of hyperactivity, increased thyroglobulin turn over, and significantly decreased plasma T16 and/or interfere triiodothyronine binding to transthyretin35. Some phthalates such as DBP and DEHP may induce thyroid hyperactivity and decrease thyroxin concentrations through modulating the transcriptional activity of NIS36.The biological mechanisms of above abservations still remain unclear. Several studies have shown that thyroid hormone receptors (TRs) may be unintended targets of manufactured chemicals. Phthalates also have been reported to have TRs antagonist activity37. However, phthalate metabolites concentrations in serum significantly correlated to those in urine, suggesting that metabolites in serum could be used as biomarkers of human exposure38. Secondly, as the present study is a pilot research, the indicators of thyroid function and phthalates exposure were limited. We did not measure thyroid peroxydase antibody which was one of the most important determinants of thyroid function. In addition, only the primary metabolites of DEP, DBP and DEHP were analyzed since pregnant women were predominantly exposed to these three types of phthalates13. Finally, the cumulative exposure risks of multiple other phthalates could not be obtained in the present study.There are some limitations in the present study. Firstly, we did not collect urine samples. Urine is the best matrix for measuring non-persistent chemicals including phthalatesOur findings indicate that serum LnMBP and LnMEHP during early pregnancy are associated with decreased maternal TT4 concentrations. Besides, it is suggested that the effects of MBP and MEHP exposure on TT4 could begin as early as at the embryonic stage.40. Briefly, this cross-sectional survey, as a part of the C-ABC study, enrolled participants in the first 20 weeks’ gestation in Ma’anshan city between November 2008 and September 2009. 1,599 women with singleton pregnancies were invited to participate at their first antenatal care visit. The response rate was 97.4% . They were required to complete a self-administered questionnaire. Meanwhile, a single blood sample was collected from each woman for the laboratory examination. Finally, 1,397 pregnant women with complete data of both THs (measured in 2010) and phthalates (measured in 2012) were enrolled in the present study.The C-ABC study was conducted to examine the delayed, cumulative and interactive effects of maternal environmental exposures on birth outcomes and children’s development. All details of in- and exclusion criteria were described in our previous studiesInformed consent was obtained from all participants in our study. The study  was approved by the Biomedicine Ethical Committee of Anhui Medical University . All the study methods were performed in accordance with Helsinki declaration II.Possible confounding variables of basic demographic characteristics were obtained, including maternal age (continuous), pre-pregnancy body mass index , gestational age (continuous), residence , educational level , family monthly income , and passive smoking . The multiple imputation method was used to impute missing values.et al., 2010) was used to analysis the serum concentration of phthalates. In brief, monoethyl phthalate (MEP) was measured through external standard method, while MBP  and MEHP were measured through isotope-labeled internal standard  method.At the prenatal visit, a single peripheral venous blood sample per woman was drawn from an antecubital vein into 10 mL glass vacuum vessels. After centrifugation, samples were aliquoted and stored at −80 °C until biochemical analyses. In the Anhui Provincial Key Laboratory of Population Health & Aristogenics , a revised isotope dilution-solid phase extraction (SPE) coupled to high-performance liquid chromatograph-tandem mass spectrometry  method . The functional sensitivity of TSH was 0.004 mIU/L. The detectable concentrations of TSH, free TAll statistical analyses were performed using the Statistical Package of Social Sciences .The phthalate monoester concentrations below the LOD were replaced with a value of LOD/√2 for statistical analyses. To investigate the combined risks of multiple phthalate exposure, we calculated a single variable “PAE” through summing up the concentrations of all metabolites by weighting their molecular weight. Here is the equation.Firstly, we statistically described the concentrations of phthalate metabolites using several selected percentiles. Furthermore, as the distributions of phthalate metabolites and FT4 and TSH were right-skewed, their concentrations were natural logarithm (Ln)-transformed for statistical analyses. Multivariable linear regression analyses were used to explore the relationships between phthalate monoesters and THs. Finally, stratification analyses were applied by gestational weeks of serum sampling time . Potential confounders which were significantly correlated to THs according to Pearson correlation or ANOVA tests were included in the adjusted statistical models.


Sitotroga cerealella, is a serious pest of stored grains worldwide. Presently, the best effective control against the moth is to disrupt the sexual communication between sexes. Sexual communication in moths includes two processes in which females produce and release pheromones from the sex pheromone gland and males detect and respond to them with a relatively sophisticated olfactory system in their antennae. To better understand these processes, we studied the ultrastructure of antennal and ovipositor sensilla of S. cerealella and determined the location of the female sex pheromone gland. Seven types of antennal sensilla were identified on both sexes: sensilla trichodea, sensilla chaetica, sensilla coeloconica, sensilla styloconica, sensilla auricillica, sensilla squamiformia and Bӧhm bristles. Of these sensilla, the sensilla trichodea were significantly more abundant on male antennae than on those of females, suggesting that these sensilla may detect the sex pheromones. On the ovipositor, only sensilla chaetica of various lengths were found. The sexual gland was an eversible sac of glandular epithelium that was situated dorsally in the intersegmental membrane between the 8th and 9th abdominal segments. These results will lead to a better understanding of mate finding with sex pheromones for this worldwide pest species.The Angoumois grain moth,  Sitotroga cerealella Olivier (Lepidoptera: Gelechiidae), is one of the most serious stored-grain pests worldwide, particularly in the tropics and warm temperate regionsS. cerealella is directly protected from chemical insecticides and causes damage in both the field and storage facilitiesThe Angoumois grain moth, Cameraria ohridellaHelicoverpa zeaHeliothis virescensC. ohridellaH. zeaH. virescensManduca sexta, the olfactory sensilla on the antennae are primarily used to identify the female sex pheromone for the location of matesAgrotis segetumCydia pomonellaS. cerealella, the primary sex pheromone component is Z, E-7, 11-hexadecadien-1-yl acetateS. cerealella postulated to be involved in the detection of sex pheromones are predominantly expressed in adult antennae and display a high binding affinity with Z, E-7, 11-hexadecadien-1-yl acetateS. cerealella have not been fully investigated.The courtship and copulation of the moth are important behaviours for the pest behavioral manipulation, and the release and reception of sex pheromones regulate the mating behaviours of female and male mothsS. cerealella were examined using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to deduce sensilla function. Additionally, using SEM, histology and gas chromatography-mass spectrometer (GC-MS), the morphology and location of the female sex pheromone gland were determined.In this study, the external morphology and ultrastructure of the sensilla on the antennae and ovipositor of S. cerealella was similar between males and females , sensilla chaetica (SC), sensilla coeloconica (SCo), sensilla styloconica (SSt), sensilla auricillica (SA), sensilla squamiformia (SSq), and Bӧhm bristles (BB). The ST, SCo, SSt, and BB also had two subtypes. The length and basal width of these sensilla are shown in S. cerealella antennae. These sensilla were subdivided into two subtypes (STI and STII) according to the surface substructure and the length  than the STI . In addiSensilla coeloconica were on the ventral side of the flagellum, and two subtypes were identified: SCoI and SCoII. SCoI had a flower-like shape with a peg in the center, which was surrounded by approximately 11–14 inwards-facing spines that resembled petals . The cutSensilla auricillica had the appearance of a rabbit ear or a new leaf of Gramineae . The cutSensilla squamiformia were distributed on the dorsal surface of all parts of the antenna and were covered with scales. These sensilla were scale-like, but were smaller and narrower than normal scales . The appSensilla styloconica were divided into two subtypes, SStI and SStII, based on specific features. SStI were on the distal margin of each flagellomere with the exception of the apical segment, and there was only one sensillum on each segment . This tyThe short spines of Bӧhm bristles are classified by Schneiderth abdominal segment of female S. cerealella was densely clothed with scales; whereas the 8th and 9th uromeres were without scales  and microtrichia were found on the ovipositor of e region . These se region . Microtre region .th and 9th segments were fully exposed . The females remain in this position for several hours to attract males. These results further verified that the terminal abdominal segments  were the source of the female sex pheromone in S. cerealella.The location of the sex pheromone-producing gland in female S. cerealella were analyzed, and the structure and location of the female sex pheromone gland were also determined. These analyses allow us to better understand the olfactory mechanisms for chemical communication between males and females and provide a useful foundation for future electrophysiological and behavioural studies.In this study, the morphology, fine structures and distributions of the sensilla on the antennae and ovipositor of S. cerealella were obtained from a laboratory colony that was originally collected from Wuhan, Hubei Province, China, in July 2010. The moths were maintained in cages (25 × 25 × 25 cm) containing an approximately 3-cm-thick layer of wheat on the bottom. To obtain virgin adults, each kernel that contained one pupa was separated in a small glass tube, which were maintained at 28 ± 1 °C, 75% ± 5% relative humidity and a photoperiod cycle of 14 h light/10 h dark. The pupae were checked daily until eclosion, and the newly emerged adults were used in the experiments.Adult S. cerealella were first anaesthetized by aether. Then, the antennae and extended ovipositors  and finally immersed in 100% isoamyl acetate for drying in a critical point drier . After mounting on a holder using double-sided adhesive tape, all samples were sputter-coated with gold/palladium (40:60) and observed using a Hitachi jsm-6390 or SU8000 scanning electron microscope.For transmission electron microscopy, the antennae were embedded in SPI-812 resin. Ultrathin sections were cut with a diamond knife on a Leica UC6 ultramicrotome, double-stained with uranyl acetate and lead citrate and finally observed on a Hitachi HT7700 transmission electron microscope operating.The ovipositors from females were fixed in 4% paraformaldehyde for 24 h. After fixing, samples were dehydrated in a graded series of ethanol: 75% for 4 h, 85% for 2 h, 90% for 2 h, 95% for 1 h, and twice at 100% for 30 min each. The specimens embedded in wax were then serial sectioned. The slices, 4 μm thick, with sagittal and cross sections, were stained with hematoxylin-eosin stain and later dehydrated. Finally, rhamsan gum was used to seal the slices, with the histology sections examined with a Nikon Eclipse Ti-SR microscope .th and 9th abdominal segments) were then excised from twenty virgin females and extracted in 100 μl of hexane for 30 min. The extracts were analyzed using GC-MS. Another twenty ovipositors from 2-d-old scotophase females were separately cut into three sections  as shown in Pressure was applied to the abdomen of virgin female moths (2-d-old) until the terminal abdominal segments extruded. Ovipositors  with a GC-MS system (7890A-5975C). The inlet (250 °C) was operated in splitless mode  and 3 μl of the sample was injected (MS solvent delay 4 min). The oven was operated as follows: 50 °C for three minutes to 230 °C for ten minutes at a rate of 20 °C/min. Helium carrier gas was used at a constant flow rate of 1.0 ml/min. Electron impact mass spectra were recorded at 70 eV ionization energy in full scan mode. The ionization source temperature was set at 230 °C.The morphological terms in this study followed those of SchneiderHow to cite this article: Ma, M. et al. Ultrastructure of sensilla of antennae and ovipositor of Sitotroga cerealella (Lepidoptera: Gelechiidae), and location of female sex pheromone gland. Sci. Rep.7, 40637; doi: 10.1038/srep40637 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Analyses of BMD and LCT-13910 genotypes revealed that mean BMD was the highest  in the individuals with lactose tolerance genotypes (TT and CT). We also found possible association of lower BMD at the radius and CC genotypes due to higher but not statistically significant frequency of osteoporosis in the lactose intolerant group (p = 0.60). Statistically significant correlation was found between BMD and females aged 20–35 years, with tendency to reduce BMD with age (p = 0.02).The relation of LCT-13910 genotypes and bone mineral density (BMD) has been the subject of modern-day human population studies, giving inconsistent results. In the present study we analyze for the first time a relation of LCT-13910 genotypes and BMD in historical skeletal individuals. Ancient population might be a model for testing this association due to elimination of non-natural factors affecting bone density. Among 22 medieval individuals from Sanok churchyard  we identified 4 individuals with osteoporosis (mean BMD = 0.468 g/cm MCM6 gene [Lactose intolerance (LI) is inherited as an autosomal recessive trait which leads to the down-regulation of lactose-phlorizin hydrolase (LPH) enzyme activity, in the intestinal cells, what decreases the ability to convert lactose into absorbable sugars of glucose and galactose after weaning . The decCM6 gene .One of the major debated issues connected with lactose is the relation of LI with bone density and the risk for fractures and osteoporosis. Although the influence of LI on bone mineral density (BMD) and calcium intake were discussed in recent decades, this issue still remains unclear. Most of clinical studies focused on postmenopausal women and elderly men. In result, several authors reported significant association of LCT genotypes and BMD –6, whileIt was reported that LCT-13910 CT and TT genotypes associated with LT were subjected in Europe to a strong positive selection for over 3000 years. The TT and CT genotypes were absent in early Neolithic Farmer populations and present in only 10% of Bronze Age European people . In EuroWe assume that ancient population might be a good model for testing association of BMD and LI, due to elimination other than natural and physiological factors which may influence bone density. Milk was one of the main sources of calcium, at the time, what could make the possible relation between LCT-13910 genotypes and BMD more prominent. Here we present for the first time the relation of LCT-13910 genotypes and BMD in ancient human population and the possible association of BMD and age of women in reproductive period.Analyses were conducted on 22 medieval individuals  from Sanok churchyard  , dated f2 according to [To determine BMD, we used fully preserved right or left radius with fused epiphyses and with no signs of soil erosion. The bones were measured by Dual X-Ray Absorptiometry (DXA) using Hologic Discovery W densitometer  at the Medical University Hospital in Wroclaw, Poland. DXA allowed to measure bone mineral content (BMC) using absorption data of two different length X-ray beams what led to determine bone mineral density (content of hydroxyapatite in bone) in g/cmrding to . This terding to . To comprding to , 18. In rding to . Norm  and LCTR (GCGCTGGCAATACAGATAAG) primers fused with barcodes and Ion Torrent adapters. For each DNA extract, 2–3 separate PCR reactions were set up using AmpliTaq Gold 360 Master Mix (Life Technologies), 0.2 μM each primer, and 3 μl of DNA template. PCR conditions were as follows: 2 min at 94°C, 40 cycles of 1 min at 94°C, 30s at 60°C, 1 min at 72°C and final extension at 72°C for 5 min. Negative controls were set up during extractions (one control for every eight extractions) and amplifications (one negative control for every eight PCR reactions). Pooled PCR products were separated by 2% agarose gel electrophoresis and purified with QIAquick® Gel Extraction Kit (Qiagen). Fragment length distribution and concentration of libraries were measured with the use of 2200 TapeStation system (Agilent). Sequencing was conducted on high-throughput PGM Ion Torrent system using Ion 314™ Chip Kit v2 and the Ion PGM Hi-Q sequencing kit v2 following the manufacturer’s instructions.Teeth or petrous bones were collected using disposable gloves and mask in order to minimize the risk of modern DNA contamination. DNA extractions were conducted in the sterile laboratory dedicated to ancient DNA analyses, at the Faculty of Biology, Adam Mickiewicz University in Poznan (AMU), Poland. Samples were cleaned with ~ 5% NaOCl, rinsed with sterile water and UV irradiated as described before . Roots oeviously . A 52 bphttp://hannonlab.cshl.edu/fastx_toolkit/) was used to separate reads by barcodes using a one mismatch threshold. Cutadapt v1.8.1 software was applied to trim the adapters [Sequencing data were processed using a custom analytical pipeline. The fastx_barcode_splitter.pl script . The differences between LI and LT groups in OP classification were tested by Fisher’s exact test. Non-parametric version of ANOVA  was used to test the heterogeneity in BMD between different genotypes . The statistical analyses were performed in Python (3.5.0) with the help of SciPy library (0.18.1) .2 to 0.683 g/cm2, with mean value of 0.560 g/cm2 (SD = 0.094) at the radius bone (2 (SD = 0.089) for males and females, respectively. Among analyzed individuals, all BMD classes  have been assigned based on T-score values. We identified 4 individuals with OP , 10 individuals with OPN  and 8 individuals with N . Using linear regression, statistically significant correlation was found between BMD and age of females (aged 20–35 years) with tendency to reduce BMD with age   .LCT-13910 genotypes were successfully determined in 13 individuals . The dis2, SD = 0.027) and LCT-13910 CT . The lowest values were observed in LCT-13910 CC genotypes . Female with the lowest BMD (sample no. 243) was found in LI group. Among LT group only one individual with OP was identified (sample no. 41), while three individuals with OP were found  among LI. According to Fisher’s exact test the distribution of OP in LI and LT was not statistically significant (p = 0.60). The heterogeneity in BMD between different genotypes , analyzed with the use of Kruskal-Wallis H test, was also not statistically significant (p = 0.73).Comparisons of BMD and LCT revealed that mean BMD was the highest in LT individuals with LCT-13910 TT genotypes (mean BMD 0.583 g/cmp = 0.60). Although there are no ancient comparative data on BMD and LCT-13910 genotypes, our data are consistent with previously reported results acquired for radius, total hip and the Ward’s triangle BMD in a present-day postmenopausal Caucasian women [This is the first study of BMD and LCT-13910 genotype relations that was performed on an ancient population. Although the number of analyzed individuals is low when compared to modern population studies, we found possible association of lower BMD and LI, at the radius. We noted higher, but not statistically significant frequency of OP in LI group p = 0.0. Althouan women . The assan women  and recean women .Among LT individuals we found one adult man with OP, aged 50–55 years (sample no. 41). The appearance of OP in this individual could be the effect of an age related bone loss or the idiopathic osteoporosis connected with some secondary cause, which is also observed in present-day patients . NotablyIn conclusion, our data from historical medieval individuals suggest possible association of LCT-13910 CC and lower BMD; however, we cannot exclude samples bias due to small population size. Past population could be treated as a model for verification of LCT-BMD association, although to get a better understanding of this relation, future studies should include more ancient individuals from different periods of time.S1 Text(DOCX)Click here for additional data file.


P = 0.035). The area under the receiver operating characteristic curve for PCT was 0.715  and was higher than that for CRP (0.598 (0.368–0.828)) or WBC counts (0.502 (0.307–0.697)). In patients undergoing TACE and/or RFA, a significantly lower number of antibiotics were prescribed during the study period than during the prestudy period (P < 0.001). In conclusion, PCT might be a biomarker for diagnosing infection and guiding antibiotic treatment to reduce unnecessary antibiotic use in patients with fever after TACE and/or RFA.This prospective observational study aimed at investigating the role of procalcitonin (PCT) in diagnosing bacterial infection and guiding antibiotic therapy for hepatocellular carcinoma (HCC) patients with fever after transarterial chemoembolization (TACE) and/or radiofrequency ablation (RFA). Ninety-seven cases (84 patients) were enrolled. Serum PCT, C-reactive protein (CRP), and white blood cell (WBC) counts were measured on the day of fever onset (day 0) and days 1, 3, 5, and 7 of fever. Empirical antibiotics were initiated only if PCT was ≥0.5 ng/mL or specific infection foci were suspected. An infectious cause was found in nine cases. PCT on day 0 of fever was significantly higher in patients with bacterial infection than in those without infection ( Transarterial chemoembolization (TACE) and radiofrequency ablation (RFA) are the most widely used primary treatments for unresectable hepatocellular carcinoma (HCC) . These pα [Procalcitonin (PCT) is a 116-amino-acid prohormone of calcitonin, which is normally synthesized in C cells of the thyroid gland. The diagnostic value of PCT to predict bacterial infection has been studied in various diseases and patient groups , 11. In α . Dabbousα . HoweverThis prospective study was performed between December 2012 and December 2013 in Chonnam National University Hwasun Hospital . All patients admitted for TACE and/or RFA to treat HCC were screened during the study period. Patients who developed fever (≥38.0°C) within 7 days after TACE and/or RFA were included. Patients with documented fever, infection, or antibiotic use within 7 days before TACE and/or RFA were excluded. To compare the amount of antibiotic use between the study period (December 2012 through December 2013) and the prestudy period (November 2011 through November 2012), all medical records of patients admitted for TACE and/or RFA in the prestudy period were retrospectively reviewed. This study was approved by the institutional review board of Chonnam National University Hwasun Hospital . Written informed consent was obtained from all participants before enrollment in this study.Preprocedural care for TACE was performed including hydration, antiemetics and antihistamine, and a single dose of dexamethasone 5 mg. Although some recommend the prophylactic antibiotics for TACE in patients with severe liver cirrhosis, none of the patients included in this study received the prophylactic antibiotics, because their Child-Pugh score is A or B , 15. DurPCT, CRP, white blood cell (WBC) counts, total bilirubin, albumin, alkaline phosphatase (ALP), aspartate aminotransferase, alanine transaminase, prothrombin time , blood urea nitrogen, creatinine, and electrolytes were measured just before TACE or RFA and on the day of fever onset (day 0). PCT was measured using an immunoluminometric assay  and the Cobas e602 analyzer (Roche) with a lower detection limit of 0.02 ng/dL. On day 0 of fever, two sets of blood culture samples were collected, and urinalysis and chest radiography were performed for all patients to evaluate the possible infection foci. Other imaging studies, ascites analysis, or culture of clinical samples such as sputum, urine, ascites or pus, and additional blood cultures were performed if indicated. An attending physician and infectious disease specialist examined each patient separately to evaluate the infection foci daily for 7 days after fever developed. We used the most widely used cutoff value for PCT of 0.5 ng/mL for the diagnosis of bacterial infection –18. If tα-fetoprotein (AFP) level is ≥200 ng/mL, typical characteristic findings of HCC on dynamic contrast enhancement computed tomography (CT) or magnetic resonance imaging (MRI) or, if the serum AFP level is <200 ng/mL, typical characteristic findings of HCC in 2 or more radiologic studies (dynamic contrast enhancement CT or MRI or hepatic artery angiography), or (2) liver cirrhosis with a tumor ≥ 2 cm and typical characteristic findings of HCC on dynamic contrast enhancement CT or MRI regardless of serum AFP level. Typical characteristic findings of HCC are defined as contrast enhancement in the arterial phase and washout in the portal/venous phase in comparison with liver parenchyma [The diagnosis of HCC was confirmed either by histological examination or by the clinical diagnosis: (1) chronic viral hepatitis infection or liver cirrhosis and, if the serum renchyma , 20. TNMrenchyma . Fever arenchyma . Infectiχ2 test as appropriate was used for comparing categorical variables. Continuous variables were analyzed using the Mann–Whitney U test. To compare the predictive value of CRP, PCT, WBC counts, and neutrophil percentage, receiver operating characteristic (ROC) curves were constructed and the area under the curve (AUC) was determined. The outcome variable was the development of bacterial infection during the first week after TACE and/or RFA. All tests of significance were two-tailed, and P values < 0.05 were considered statistically significant. The Statistical Package for the Social Sciences  was used for statistical analyses and graphics.Discrete variables are expressed as frequency (percentage) and continuous variables as means and standard deviations or median and interquartile ranges (IQRs) according to their homogeneity and distribution. Fisher's exact test or the Pearson Of the 563 TACE and/or RFA procedures performed during the study period, 97 cases (84 patients) of fever after TACE and/or RFA were included in this study. An infectious cause was found in nine (9.3%) of these cases, but no infectious cause was found in the other 88 cases (90.7%). The clinical characteristics and status of HCC showed no statistical differences between the two groups .The mean duration of fever was 1.2 days ranging from 1 to 8 days. For each patient with or without bacterial infection, the median duration of fever was 1.0 day (ranging from 1 to 8 days) and 1.0 (ranging from 1 to 6 days). Most of the patients (94 of 97 patients) developed the fever within 3 days after TACE and/or RFA. The overall median of peak body temperature was 38.1°C ranging from 38.0 to 39.8. For each patient with or without bacterial infection, the median of peak body temperature was 38.0°C (ranging from 38.0 to 39.8) and 38.1°C (ranging from 38.0 to 39.2). There was no statistical difference in peak body temperature and duration of fever between patients with and without bacterial infection.Antibiotics were prescribed in 25 cases . The median duration of antibiotic therapy was 8.0 days (IQR 4.5–13.0), 13.0 days (IQR 5–18) in patients with bacterial infection, and 6.0 days (IQR 2–10) in patients without bacterial infection.Of the nine cases of proven bacterial infection, hepatobiliary infection such as cholecystitis, liver abscess, or cholangitis was the most common (three cases) followed by urinary tract infection (UTI) and pneumonia (two cases each) and RFA site cutaneous abscess and bacteremia with unknown primary focus (one case each) .P = 0.035). However, there were no significant differences in WBC counts, neutrophil percentage, or CRP level between patients with and without bacterial infection. ALP and total bilirubin levels were also higher in patients with bacterial infection than in those without infection   than in those without infectious etiology   .P < 0.05), but there was no significant difference at 7 days of fever (median ng/dL (IQR), 0.17 (0.16 ~ 0.38) versus 0.15 (0.12–0.93), P = 0.556). There was no significant difference in the median CRP level between the two groups.The kinetics of PCT and CRP over 7 days after fever are shown in The AUC ) of PCT on day 0 of fever was 0.715 (0.538–0.892), which was higher than that of CRP (0.598 (0.368–0.828)), WBC counts (0.502 (0.307–0.697)), and neutrophil percentage (0.647 (0.445–0.849)) . When thP < 0.001). There was no difference in the incidence of infectious complications between the prestudy and study period. The average duration of antibiotics prescribed and length of hospital stay were also significantly lower during the study period   .This study showed the usefulness of PCT for making a decision regarding administration of antibiotics for patients who develop a fever after undergoing TACE and/or RFA for HCC. The accuracy of PCT measured on day 0 of fever was significantly higher, with an AUC value of 0.715, than that of other inflammatory markers such as CRP and WBC counts to discriminate a fever related to bacterial infection from a noninfectious fever. In addition, consecutively measured PCT levels on days 0, 1, 3, and 5 of fever were significantly higher in patients with bacterial infection than in those without infection. In our study, we used PCT level as a guide for determining whether or not to start antibiotic therapy. When we compared the total amount and duration of antibiotic prescriptions in patients with fever after TACE and/or RFA between the prestudy and study period, we found that a significantly lower number of patients were prescribed antibiotics in the study period than in the prestudy period. The average duration of antibiotic use and length of hospital stay were also decreased in the study period. There was no difference in the incidence of infectious complications between the prestudy and study period.TACE and RFA are generally well-tolerated procedures for treating HCC. However, a considerable number of patients experience the postembolization or postablation syndrome, which is a transient self-limiting complex of symptoms or signs of fever and general malaise , 22. In E. coli bacteremia, 2 cases of UTI, and 2 cases of pneumonia. However, it is hard to exclude pneumonia and UTI that developed within 7 days from procedure-related infections. Moreover, five cases of hepatobiliary or GI infections also cannot be concluded as procedure related, because such infections are commonly occurring in patients with liver cirrhosis and/or HCC without TACE/RFA. For these reasons, we included all cases with bacterial infections developed within 7 days after procedure. The overall rate for infectious complication was 1.6% (9/563 cases), which is comparable to previous study [We had five cases of hepatobiliary or gastrointestinal (GI) infections including primary us study .In previous studies, PCT has been shown to be useful as a diagnostic marker for bacterial infection in patients with cirrhosis , 24. HowIn this study, antibiotics were prescribed only when PCT was ≥0.5 ng/mL or a suspicious infection focus was present. As antibiotics had been empirically prescribed in patients with fever after TACE and/or RFA before the study period in our institute, we compared the total amount and duration of antibiotic use in patients with fever after TACE and/or RFA between the prestudy and study period. We found that a significantly lower number of patients were prescribed antibiotics in the study period than in the prestudy period. The average duration of antibiotic use was also decreased in the study period. There was no difference in the incidence of infectious complications between the prestudy and study period. Many institutes still prescribe antibiotics routinely to patients with fever after TACE or RFA , 26. SomThis study has some limitations. First, as this study was performed in a single center and included only nine cases of bacterial infection, further extensive research is warranted before applying these results generally. Second, a PCT level of <0.5 ng/mL did not have 100% negative predictive value, and two patients with bacterial infection showed a low PCT level (<0.5 ng/mL) during 7 days of follow-up after fever onset. However, even in these cases, the administration of empirical antibiotics was not delayed as other tests such as chest radiography, a physical examination, and a microbiologic study showed evidence of bacterial infection. As there is no biomarker with 100% sensitivity or specificity for the diagnosis of infection, a physician's careful evaluation and other laboratory and microbiological tests should always be conducted at the same time.In conclusion, PCT might be a biomarker to detect infectious complications after TACE and/or RFA in HCC patients, if physical examination and laboratory and microbiological tests are warranted to detect infection. PCT-guided antibiotic treatment could contribute to reduce unnecessary use of antibiotics without worsening outcomes in patients with fever after TACE and/or RFA.


Nymphon australe, with an apparently large population size compared with other sea spider species, is an ideal target to look for molecular signatures of past climatic events. We analysed mitochondrial DNA of specimens collected from the Antarctic continent and two Antarctic islands (AI) to infer past population processes and understand current genetic structure. Demographic history analyses suggest populations survived in refugia during the Last Glacial Maximum. The high genetic diversity found in the Antarctic Peninsula and East Antarctic (EA) seems related to multiple demographic contraction–expansion events associated with deep-sea refugia, while the low genetic diversity in the Weddell Sea points to a more recent expansion from a shelf refugium. We suggest the genetic structure of N. australe from AI reflects recent colonization from the continent. At a local level, EA populations reveal generally low genetic differentiation, geographically and bathymetrically, suggesting limited restrictions to dispersal. Results highlight regional differences in demographic histories and how these relate to the variation in intensity of glaciation–deglaciation events around Antarctica, critical for the study of local evolutionary processes. These are valuable data for understanding the remarkable success of Antarctic pycnogonids, and how environmental changes have shaped the evolution and diversification of Southern Ocean benthic biodiversity.The evolutionary history of Antarctic organisms is becoming increasingly important to understand and manage population trajectories under rapid environmental change. The Antarctic sea spider  This cosis spp. ,24, Austs, 1902) , Colosseeis spp. ,26, and sing COI ,27.Nymphon australe Hodgson 1902 is the most abundant and frequently collected SO species with wide bathymetric (8–4136 m depth) and geographical ranges. It has been recorded from around the Antarctic continent, the southern tips of South America, South Africa and New Zealand, although New Zealand representatives are considered a subspecies N. australe caecum Gordon, 1944 [N. australe is a single widespread species with intraspecific variation of some characters sometimes overlapping interspecific differentiation [N. australe, based on 57 specimens from the Antarctic Peninsula (AP) [N. australe as a true circumpolar species with distinct genetic differentiation among individuals from different localities and regions.on, 1944 ,28; in getric 8–46 m depthula (AP)  and lateetric 8–46 m depthN. australe populations around Antarctica to understand the wide geographical distribution proposed for the species, and (ii) relate the genetic structure observed with historic events including refugia survival during LGM and population expansion during deglaciations. We examined the genetic structure of the species at a range of spatial (1000s km to 100s km) and bathymetric scales taking advantage of: (i) large geographical coverage of the dataset, one of the largest for benthic SO invertebrates; (ii) wide bathymetric range with individuals collected from 25 to 1260 m depth; and (iii) the unusual and relatively large number of individuals collected at several locations in the EA.The present study aimed to: (i) investigate the genetic differentiation and demographic histories of 2.2.1.N. australe specimens collected by a variety of sampling means from diving to trawling at 69 sites in the SO between 2007 and 2011  aiming to collect, determine and curate well-preserved SO benthic fauna . Expeditn et al.  and Aran [et al. . Sequenc2.2.et al. [p-distance ≤ 1%), discouraging the use of this ITS fragment for within-species level analyses in N. australe. The use of microsatellites to complement this COI dataset was also attempted; however, the developed markers were too conserved. Four markers were monomorphic for all analysed samples with half of them showing no polymorphism even across species boundaries [Most of the samples were submitted to the Canadian Centre for DNA Barcoding as part of the barcoding agreement between CAML  and the et al. . Other get al.  and the et al.  showed vundaries .Nymphon mendosum , an Antarctic species closely related to N. australe [p-distance mean among N. australe sequences was 0.01 while between N. australe and N. mendosum was 0.08. A haplotype parsimony network was constructed using TCS 1.21 [rBayes 3.1.2 [rModelTest v.2 [Uncorrected pairwise distances among the COI sequences were calculated with 1000 bootstrap replicates under a J–K model in MEGA v. 7 . Nymphonaustrale , was incTCS 1.21  with 95%es 3.1.2  using MrTest v.2 . Based oTest v.2 , the HKY2.3.N. australe was measured for each SO region and for the subregions within the EA using DNASP v. 5.00.7 [FST = 0) among SO regions and within the EA, and the corrected p-values for population differentiation among pairs of populations were tested with Arlequin v. 3.0.1 [Based on the sampling localities, COI sequences were grouped in one of four main SO geographical regions: Antarctic Peninsula (AP), East Antarctic (EA), Weddell Sea (WS) and Antarctic Islands (AI). Furthermore, the EA was divided into four subregions: Terre Adélie (TA), Ross Sea (RS), Davis Station (DS) and Bruce Rise (BR). Within the AI, there were two subregions: Bouvet Island (BI) and South Sandwich Islands (SSI) .To infer the spatial genetic structure of v. 2.0.0  in the Rv. 2.0.0 ). The so2.4.N. australe at various spatial and temporal scales related to glacial refugia [D and Fu's SF) were run in DnaSP to estimate deviations from the mutation-drift equilibrium and infer past population processes. The observed distribution of pairwise differences between sequences  was examined in Arlequin under a model of population expansion and the resulting shape of the distribution was compared to a simulated dataset under a spatial expansion model [To detect demographic changes such as population expansion or contraction in  refugia , the numon model ,44 and aon model . Signifiluctuate 1.4 [g) and the θ parameter , the initial value of θ was estimated using the approach of Watterson [We used the coalescent-based method implemented in Fuate 1.4  to estimatterson . All runT) for the analysed COI fragment based on T = τ/(2µk), where τ is the expansion time estimator, µ the mutation rate and k the sequence length [θ0 = 2N0u (before population growth/decline) and θ1 = 2N1u (after the population growth/decline). Here, we apply a 10-fold correction when estimating N. australe population expansion times, as it has been suggested that the short-term mutation rate is 10-fold higher than long-term substitution rates [Molecular clock estimates were used to approximately date the timing of population expansions as in Rogers , calculae length . The demon rates ,51.3.3.1.N. australe had a length of 554 bp and represented 85 unique haplotypes  of which 10 were common haplotypes , while the majority were uncommon haplotypes with only one or two representatives .The final COI alignment of 364 sequences of ntatives . We detek) was lowest in AI (k = 6) and highest in EA (k = 50), although this was probably related to the different sampling effort in each locality given that haplotype diversity was similar in these two locations . Genetic variation was low in the WS, which had around half the haplotypic diversity of the other localities, as well as the lowest average number of nucleotide differences (Π) and mean nucleotide diversity (π). The number and the proportion of private haplotypes were lowest in AI, although this was the region with the smallest sample size . COI pairwise FST relationships and significance comparisons supported the structuring of SO regions , while FST values between EA and AI samples were the lowest, but still significantly greater than zero (FST = 0.10).There was significant genetic differentiation among the four regions sampled around Antarctica . Most haplotypes were found only within a single region except for the AI where a large proportion of haplotypes were found elsewhere. Furthermore, the haplotype network had a predominant star-like pattern at the regional level, particularly for EA (b). Bayesian models computed with Geneland detected three main clusters , cluster B grouped all EA individuals with the SSI (b), and cluster C included all AP individuals (c). Values of cluster membership were approximately P = 0.65 for each cluster locality.A Bayesian phylogenetic analysis did not show segregated, well-supported clades related to region; however, the COI haplotype network coupled with the Bayesian consensus while supporting plotypes a. Most hy for EA b. Bayesis K = 3, : clusters K = 3, a, cluste the SSI b, and clividuals c. ValuesFST results within the AP and WS indicated low differentiation within regions, over 90% of the variation was owing to the differences within populations. Within the EA region, there was significant genetic differentiation among the five subregions sampled , although this was largely a result of the large and significant differences between DS samples and those from all other subregions (FST < 0.025). Bayesian cluster analysis revealed two clusters . Samples from DS were all collected in about 25 m depth, whereas most other EA samples were collected deeper than 200 m. A subset of the TA samples collected by the REVOLTA program from approximately 40 m depth showed no evidence of significant genetic differentiation from deeper TA samples . The AI populations (BI and SSI) were also significantly different , while the WS did not show significant differences  between the easternmost and westernmost populations. Genetic differentiation within AP was significant owing to differences between northeastern and southernmost populations of the AP  seem to indicate that N. australe populations have gone through relatively recent expansions. We obtained a similar pattern in separate analyses of the WS, EA and AP data. Either an L-shaped or a unimodal distribution of pairwise differences was obtained for each region, supporting the existence of a demographic expansion in each region; this pattern was corroborated by the negative and significant Fu's SF test values and the non-significant goodness-of-fit tests. Tajima's D was not significant (p > 0.05) for AP and EA, suggesting there has not been historical reduction in effective population size in these regions  showed non-significant neutrality tests, rejecting the population expansion model  (g = 2187 (s.d. = 170.970) in EA to g = 8151 (s.d. = 403.155) in WS (The on model . Althougctively) . However5) in WS .Sudden growth model analyses detected a population expansion at about 15 thousand years before present (kyr BP) at EA  and an older expansion at the AP (21.2 kyr BP). These estimates (obtained after applying the 10-fold correction) are in agreement with the high genetic diversity calculated for each of the two regions. On the other hand, WS and AI populations with lower values of haplotype and nucleotide diversity seem to have gone through more recent population expansions around 4.3 and 5.9 kyr BP, respectively .Figure 4.N. australe is comprised of regionally distinct populations that appear to have undergone recent population expansion. Here, we show: (i) further evidence to confirm that N. australe, a benthic brooder, is indeed circum-Antarctic; (ii) that the AP and EA populations are genetically more diverse suggesting multiple demographic contraction–expansion events possibly associated with deep-sea refugia, while the low genetic diversity at the WS points to a more recent expansion possibly of shelf ice-free refugia; (iii) N. australe seems to have undergone demographic expansions during the mid-Pleistocene (15–21.2 kyr BP), suggesting multiple LGM refugia; (iv) support for population expansion in the AI associated with colonization from the continent, and (v) no genetic boundaries detected in East Antarctica, except for the clear segregation of samples from the vicinity of DS, an area particularly isolated from the more open habitats sampled off TA.The circum-Antarctic sea spider, 4.1.N. australe as a circum-Antarctic species [N. australe individuals from across Antarctica that together formed a single haplotype network precluding any suggestion of cryptic speciation  within the species. Although the use of a single mitochondrial marker is often considered limiting in the context of detecting recent speciation and inferring spatial genetic structure [N. australe.Our study supports previous work proposing  species . Here, wtructure ,52, our tructure  and ITS tructure ,31. ThusColossendeis megalonyx Hoek, 1881 [Pallenopsis patagonica  [N. australe is thus surprising, and raises questions on how to reconcile a wide distribution, more characteristic of a species with pelagic dispersal, with the life-history traits of the species  ,24); simk, 1881) –57; Amphk, 1881) ,59; Ostrk, 1881) , Nudibrak, 1881) ; Crinoidk, 1881) ,62). By k, 1881) ,64. The the male ). The roale FST, . Unfortu4.2.N. australe  is higher than that for other invertebrates distributed throughout Antarctica including echinoderms [et al. [A. cornigera, in which distant populations on the Antarctic continental shelf (WS and TA) clustered together while the islands segregated as distinct clusters [et al. argue that A. cornigera dispersal is limited by the ACC acting as major dispersal barrier, and by a relatively restricted depth range (max of 1180 m) limiting access to the deep sea. Here, the results could be seen as support for the hypothesis of connectivity between the SO islands and the continent [N. australe (known max depth 4136 m); however, the large and significant FST values found indicate regions are not connected. An alternate explanation for the grouping of samples from the islands with those from the continent is that these geographical populations are isolated and are in the process of diverging, but owing to post-LGM colonization, ancestral genetic signatures might remain.The genetic diversity estimated for inoderms  and arthinoderms  in whichinoderms . The obs [et al.  for the  [et al.  and Antaclusters . Dömel eontinent ,26 being4.3.N. australe populations, as corrected estimates (instead of 40–220 kyr BP with no correction applied) fit well with accepted hypotheses of benthic invertebrate refugia survival and population expansions associated with deglaciation events after the LGM about 20 kyr BP [N. australe populations expansion , as welturqueti ).ex situ, on the shelf of neighbouring continents or sub-AI [in situ on the continental slope and deeper waters of the SO [in situ in shelf refugia [in situ. Our N. australe findings agree with those views, but, different hypotheses apply (2 and 3 above) depending on the region.There is strong evidence that, during the last glaciations, ice sheets extended to cover the continental shelves of Antarctica ; howeverr sub-AI ,77; (ii)f the SO ; and . These time estimates of expansion, 15.5 kyr BP at EA (13.6 excluding the differentiated DS samples), and 21.1 kyr BP at the AP, fit with the dating of post-LGM deglaciation events in these regions . It BP in AP .N. australe populations from EA and AP are genetically diverse possibly owing to repeated colonization events from shallow to deep and deep to shallow and changing landscape owing to ice expansion and retraction, supporting the hypothesis of multiple independent glacial refugia [N. australe could be expected to retain high levels of genetic diversity [It is plausible to assume that  refugia . Also, eiversity ,27,63, diversity .N. australe from the WS are rather low. The strongly reduced diversity, lower estimates of theta and higher growth rates  detected in WS populations compared to EA and AP  belong to a single haplotype that is also the most common in the SSI [N. australe results support the notion of a recent, ‘founder effect’ type of colonization of BI from the WS, rather than a potential insular refugium, a hypothesis already proposed for a crinoid species [The genetic signal from samples collected from BI and SSI AI in  shows a  the SSI . The N.  species . This sc species ,16,27,77N. australe. Effect of homoplasy in an expanding population is a possibility, but also animals being transported from EA by oceanic hitchhiking or rafting, or by drifting on the ACC has been proposed [N. australe, this event seems relatively recent as haplotypes from SSI are mostly shared with EA haplotypes  reveal a lack of geographical structure within the EA. Low FST estimates among localities and Bayesian clustering corroborated a single group , except for the DS samples. In contrast to the AP with its complex topography seemingly facilitating the heterogeneity of populations within the region [N. australe. On the contrary, species restricted to shallow waters might form distinct faunistic communities on rocks and algae [Orchomenella [The iversity . The mosiversity b reveal e region ,27, the e region . It is le region  as is thnd algae  and may omenella . The sliomenella  or movem2 situated in the Vestfold Hills (see www.antarctica.gov.au). Segregation may be attributed either to a depth constriction (a maximum depth of 25 m) or to geographical isolation. This genetic differentiation of the DS samples from the remaining EA individuals is also found in other studies [b), probably because all samples were taken from a relatively small area by SCUBA.Pycnogonids from DS seem to represent the most genetically distinct population. DS is enclosed in a well-sheltered ice-free bay of 400 km studies ,58 and mN. australe genetic structure within EA suggest that the eurybathic condition of the species has allowed gene flow over extensive areas of EA, as there is no evidence of bathymetric constraint for the migration of this species. Individuals collected by the REVOLTA program from TA at 40 m depth clustered with deeper waters samples from EA collected between 200 and 1230 m.Clearly, N. australe is one of the Antarctic brooding invertebrates with the widest distribution. The species shows different demographic histories depending on the Antarctic region, possibly shaped by the characteristics of the deglaciation events post-LGM. The study reinforces the notion of the strong effect of climatic events and environmental conditions on the patterns of diversity and structure in Antarctic benthic fauna. Nymphon australe is a key species for the understanding of microevolutionary forces that could reveal connectivity and dispersal mechanisms and demographic processes in the SO.In conclusion, our results confirm that